System and method for monitoring a reaction within a receptacle vessel

ABSTRACT

A system for monitoring reactions with a plurality of receptacle vessels that includes: an incubator; a movable receptacle carrier contained within a temperature-controlled chamber of the incubator; one or more fixed fluorometers configured to measure a fluorescent emission and positioned with respect to the receptacle carrier to measure fluorescent emissions from receptacle vessels carried on the receptacle carrier into an operative position with respect to each fluorometer; one or more fluorescent reference standards mounted on the receptacle carrier; and a controller configured to control operation of the receptacle carrier and the one or more fluorometers to determine if a fluorescent emission intensity of one or more of the fluorescent reference standards deviates from an expected fluorescent emission intensity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/902,657, filed Feb. 22, 2018, now U.S. Pat. No. 10,732,112, issuedAug. 4, 2020, which is a divisional of U.S. application Ser. No.13/912,525, filed Jun. 7, 2013, now U.S. Pat. No. 9,945,780, issued Apr.17, 2018, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/659,590, filed Jun. 14, 2012, thedisclosure of each of which is hereby incorporated by reference.

FIELD

The present invention relates to systems and methods for detectingfailure or deteriorated performance of an optical signal detector, suchas a fluorometer, and, in particular, systems and methods employingfluorescent materials carried on an instrument within which thefluorometer is employed to detect fluorescent signals emitted by samplematerials.

BACKGROUND

None of the references described or referred to herein are admitted tobe prior art to the claimed invention.

Diagnostic assays are widely used in clinical diagnosis and healthscience research to detect or quantify the presence or amount ofbiological antigens, cell abnormalities, disease states, anddisease-associated pathogens, including parasites, fungi, bacteria andviruses present in a host organism or sample. Where a diagnostic assaypermits quantification, practitioners may be better able to calculatethe extent of infection or disease and to determine the state of adisease over time. Diagnostic assays are frequently focused on thedetection of chemicals, proteins, polysaccharides, nucleic acids,biopolymers, cells, or tissue of interest. A variety of assays may beemployed to detect these diagnostic indicators.

Detection of a targeted nucleic acid sequence frequently requires theuse of a probe having a nucleotide base sequence that is substantiallycomplementary to the targeted sequence or its amplicon. Under selectiveassay conditions, the probe will hybridize to the targeted sequence orits amplicon in a manner permitting a practitioner to detect thepresence of the targeted sequence in a sample. Probes may include, forexample, a label capable of detection, where the label is, for example,a radiolabel, a fluorophore or fluorescent dye, biotin, an enzyme or achemiluminescent compound.

Because the probe hybridizes to the targeted sequence or its amplicon ina manner permitting detection of a signal indicating the presence of thetargeted sequence in a sample, the strength of the signal isproportional to the amount of target sequence or its amplicon that ispresent. Accordingly, by periodically measuring, during theamplification process, a signal indicative of the presence of amplicon,the growth of amplicon overtime can be detected. Based on the datacollected during this “real-time” monitoring of the amplificationprocess, the amount of the target nucleic acid that was originally inthe sample can be ascertained. To detect different nucleic acids ofinterest in a single assay, different probes configured to hybridize todifferent nucleic acids and to emit detectibly different signals can beused. For example, different probes configured to hybridize to differenttargets can be formulated with fluorophores that fluoresce at apredetermined wavelength (i.e., color) when exposed to excitation lightof a prescribed excitation wavelength. Assays for detecting differenttarget nucleic acids can be performed in parallel by alternatelyexposing the sample material to different excitation wavelengths anddetecting the level of fluorescence at the wavelength of interestcorresponding to the probe for each target nucleic acid during thereal-time monitoring process. Parallel processing can be performed usingdifferent signal detecting devices constructed and arranged toperiodically measure signal emissions during the amplification process,and with different signal detecting devices being configured to generateexcitation signals of different wavelengths and to measure emissionsignals of different wavelengths. Suitable signal detecting devicesinclude fluorometers, such as the fluorometer described below. Oneembodiment of an automated nucleic acid diagnostic instrument isconfigured to process numerous samples carried in multiple receptacles,and each fluorometer is configured to take fluorometric readings fromthe receptacles as they are indexed past the fluorometer, for example,once every 2 seconds. Thus, 1800 times for each hour of operation of theinstrument, each fluorometer generates an excitation signal that isdirected at the sample receptacle and measures the emission signalemitted by the contents of the receptacle, generating an electricalsignal that is proportional to the intensity of the emission signal. Amalfunction (device failure and/or deteriorated performance) by afluorometer during operation of the instrument will cause errors in thefluorometric readings generated by that fluorometer and thereby causeerrors in the diagnostic results. Such malfunctions may be due tomechanical and/or electrical failures that occur during operation of thefluorometer. While the operation of the fluorometers can be checkedduring routine maintenance of the instrument, such opportunities fortesting are rare, since the testing can only be performed when theinstrument is shut down. Ideally, the instrument is operatedcontinuously for extended periods of time for maximum throughput.Therefore, it becomes impractical and non-cost-effective to repeatedlyshut the instrument down to perform fluorometer functionality testing.Accordingly, a need exists for means and methodologies for periodicallyconfirming the proper functionality of the fluorometers during theoperation of the nucleic acid diagnostic instrument.

SUMMARY

The present invention provides systems and methods for self-checking anoptical signal detector, such as a fluorometer, to detect failure ordeteriorated performance of the signal detector, wherein self-checkingcan be performed during normal use of the detector in a dynamicinstrument in which the detector is employed and without requiring thatthe detector be removed from the instrument or that operation of theinstrument be interrupted.

Aspects of the invention are embodied in a system for monitoring theperformance of a fluorometer in a dynamic environment. The systemincludes a fluorometer, a support comprising two or more fluorescentstandards, and a drive mechanism. The fluorometer comprises two or morechannels, each channel includes a separate light source, an opticalfocus and filter assembly, and an optical signal detector, and eachchannel is configured to focus the light source at a detection zone.Each fluorescent reference standard of the support corresponds to asingle channel of the fluorometer, and the support is arranged toaccommodate two or more removable reaction vessels. The drive mechanismis configured to adjust the relative horizontal positioning between thereference standards and the fluorometer such that each of the two ormore fluorescent reference standards can be positioned in or out ofoptical communication with its corresponding channel of the fluorometer.

Aspects of the invention are further embodied in a method of monitoringfluorometer performance in a dynamic system comprised of (a) afluorometer comprising two or more channels, each channel having aseparate light source, optical focus and filter assembly, and opticalsignal detector, and wherein each channel is configured to focus thelight source at a detection zone, (b) a support comprising two or morefluorescent reference standards, each fluorescent reference standardcorresponding to a single channel of the fluorometer, wherein thesupport is arranged to accommodate two or more removable receptaclevessels, and (c) a drive mechanism configured to adjust the relativehorizontal positioning between the reference standards and thefluorometer such that each of the two or more fluorescent referencestandards can be positioned in or out of optical communication with itscorresponding channel of the fluorometer. The method comprises the stepsof moving the support with respect to the fluorometer with the drivemechanism to position each of the two or more fluorescent referencestandards into optical communication with the corresponding channel ofthe fluorometer; and using the fluorescent reference standard formonitoring the performance of the fluorometer.

According to further aspects of embodiments of the invention, thefluorescent reference standard is positioned in optical communicationwith the corresponding channel of the fluorometer and out of focusrelative to the detection zone.

According to further aspects of embodiments of the invention, the two ormore fluorescent reference standards are positioned in a lineararrangement on the support.

According to further aspects of embodiments of the invention, thesupport comprises two or more linear arrangements of two or morefluorescent reference standards. Each linear arrangement may comprise aset of fluorescent reference standards having emission characteristicsthat differ from each of the other linear arrangements of fluorescentreference standards. Furthermore, one or more of the fluorescentreference standards in the linear arrangement may have emissioncharacteristics that differ from one or more other fluorescent referencestandards in the linear arrangement, and adjacent fluorescent referencestandards in the linear arrangement may have different emissioncharacteristics. The support may comprise three linear arrangements offluorescent reference standards, and at least one of the lineararrangements may comprise a set of fluorescent reference standardshaving emission characteristics that differ from the two other lineararrangements of fluorescent reference standards.

According to further aspects of embodiments of the invention, each ofthe fluorescent reference standard is comprised of fluorescent plastic,and the fluorescent reference standard may be pink, green, blue, oramber plastic.

According to further aspects of embodiments of the invention, only oneof the two or more fluorescent reference standards can be positioned inoptical communication with one of the two or more channels of thefluorometer at a time.

According to further aspects of embodiments of the invention, thefluorescent reference standard may be positioned between about 1% to 99%closer to its corresponding channel relative to the detection zone,between about 20% to 80% closer to its corresponding channel relative tothe detection zone, or between about 60% to 90% closer to itscorresponding channel relative to the detection zone.

According to further aspects of embodiments of the invention, thefluorescent reference standard may be positioned between about 1% to 99%further from its corresponding channel relative to the detection zone,between about 20% to 80% further from its corresponding channel relativeto the detection zone, or between about 60% to 90% further from itscorresponding channel relative to the detection zone. In anotherembodiment, the fluorescent reference standard is positioned at the samedistance from its corresponding channel as the distance between thechannel and the detection zone.

According to further aspects of embodiments of the invention thefluorometer is stationary, and the drive mechanism is configured toadjust the relative horizontal positioning between the referencestandards and the fluorometer by adjusting the horizontal positioning ofthe support.

According to further aspects of embodiments of the invention, thesupport comprises a rotatable carousel, the fluorometer is fixed withrespect to the carousel, and the drive mechanism is configured to adjustthe relative horizontal positioning between the reference standards andthe fluorometer by effecting angular movement of the carousel around acentral axis. The drive mechanism may comprise a motor and a drive beltconfigured to transfer rotational motion from a drive shaft of the motorto the carousel. The fluorescent reference standards maybe positioned onan outer surface of the carousel and may be embedded in an outer surfaceof the carousel. According to further aspects, the carousel comprises acircular disk having a center corresponding to the central axis and aplurality of spokes extending outwardly relative to the central axis,and the fluorescent reference standards are located on one or more ofthe spokes. The spokes may be in a non-radial orientation with respectto the central axis.

Further aspects of the invention include two or more fluorometers. Eachfluorometer comprises two or more channels, each channel includes aseparate light source, an optical focus and filter assembly, and anoptical signal detector, and each channel is configured to focus thelight source at a detection zone. Each fluorometer may have a differentlight source, optical focus and filter assembly, and optical signaldetector such that each fluorometer emits a different excitation signaland detects a different emission signal.

According to further aspects of embodiments of the invention, eachchannel of the fluorometer has a different light source, optical focusand filter assembly, and optical signal detector such that eachfluorometer emits a different excitation signal and detects a differentemission signal.

According to further aspects of embodiments of the invention, thechannels of the fluorometer are arranged in an alternating manner suchthat adjacent channels emits different excitation signals and detectdifferent emission signals.

According to further aspects of the invention, using the fluorescentstandard for monitoring the performance of the fluorometer comprisesmeasuring the intensity of the fluorescent emission of the fluorescentreference standard and comparing the measured intensity to apredetermined baseline fluorescent intensity of the fluorescentreference standard for that fluorometer.

According to further aspects of embodiments of the invention, only onefluorescent reference standard is in optical communication with itscorresponding channel at a time.

According to further aspects of embodiments of the invention, the systemfurther comprises two or more receptacle vessels containing reactionmaterials, and the method further comprises monitoring the progress of areaction occurring in each receptacle vessel with the fluorometer.

According to further aspects of embodiments of the invention, monitoringthe performance of the fluorometer occurs in sequence with monitoringthe progress of the reaction occurring in each of the two or morereceptacle vessels.

Further aspects of the invention are embodied in a system for monitoringreactions within a plurality of receptacle vessels. The system includesan incubator having a temperature-controlled chamber, a movablereceptacle carrier disposed within the temperature-controlled chamber,one or more fixed fluorometers, one or more fluorescent referencestandards, and a controller. The receptacle carrier is configured tocarry a plurality of receptacle vessels and to move the receptaclevessels within the temperature-controlled chamber. Each fluorometer isconfigured to measure a fluorescent emission and is positioned withrespect to the receptacle carrier to measure fluorescent emissions fromreceptacle vessels carried on the receptacle carrier into an operativeposition with respect to each fluorometer. The fluorescent referencestandards are mounted on the receptacle carrier. The controller isconfigured to control operation of the receptacle carrier and the one ormore fluorometers. The controller is configured to move the receptaclecarrier with respect to the one or more fluorometers to place areceptacle vessel into an operative position with respect to eachfluorometer. The controller then activates each fluorometer to measurethe fluorescent emission intensity from the sample contained in thereceptacle vessel that is in the operative position with respect to thefluorometer and determines a characteristic of the reaction based on themeasured fluorescent emission intensity from the sample contained in thereceptacle vessel. The controller then moves the receptacle carrier withrespect to the one or more fluorometers to place a fluorescent standardinto optical communication with at least one fluorometer and activatesthe fluorometer to measure the fluorescent emission intensity of thefluorescent standard that is in optical communication with thefluorometer. The controller then determines a deviation of the measuredfluorescent emission intensity of the fluorescent standard from anexpected fluorescent emission intensity. If the deviation exceeds athreshold, the controller generates an error signal, and if thedeviation does not exceed a threshold, the controller continuesoperation of the instrument.

Furthermore in an instrument configured to determine a characteristic ofa sample from the intensity of a fluorescent emission from the sample,wherein the sample is contained in a receptacle vessel that is carriedon a movable receptacle carrier, and the intensity of the fluorescentemission is measured by a fluorometer that is fixed with respect to thereceptacle carrier and is constructed and arranged to measure theintensity of fluorescent emission from a sample contained in areceptacle vessel that is moved by the receptacle carrier into adetection zone with respect to the fluorometer, further aspects of theinvention are embodied in an automated method for detecting failure ordeteriorated performance of the fluorometer with a fluorescent referencestandard mounted on the receptacle carrier. The receptacle carrier ismoved with respect to the fluorometer to periodically place thereceptacle vessel into the detection zone of the fluorometer, and aplurality of measurements of the intensity of the fluorescent emissionfrom the sample contained in the receptacle vessel are taken with thefluorometer. After taking a plurality of measurements, the receptaclecarrier is moved with respect to the fluorometer to place thefluorescent reference standard into optical communication with thefluorometer, and a test measurement of the emission intensity of thefluorescent reference standard is taken with the fluorometer. Adeviation of the test measurement from a predetermined baseline emissionintensity of the fluorescent reference standard is determined. If thedeviation determined exceeds a threshold, an error signal is generated.If the deviation does not exceed the threshold, operation of theinstrument is continued by repeating the steps of taking a plurality ofmeasurements from a sample, taking a test measurement of the fluorescentreference standard, and determining whether a deviation between the testmeasurement and the baseline exceeds the threshold until a stopcondition is reached.

According to a further aspect of the invention, determining the baselineemission intensity comprises the steps of moving the receptacle carrierwith respect to the fluorometer to place the fluorescent referencestandard into optical communication with the fluorometer before taking aplurality of measurements of fluorescent emission from samples, takingan initial measurement of the fluorescent emission intensity of thefluorescent reference standard with the fluorometer, and storing theinitial measurement as the predetermined baseline emission intensity.

These and other features, aspects, and advantages of the presentinvention will become apparent to those skilled in the art afterconsidering the following detailed description, appended claims andaccompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a reaction receptacle in the form of amultiple receptacle device unit employed in combination with anapparatus embodying aspects of the present invention.

FIG. 2 is an enlarged bottom view of a portion of the multiplereceptacle device, viewed in the direction of arrow “60” in FIG. 1.

FIG. 3 is an exploded perspective view of an incubator configured tohold a plurality of receptacles while subjecting the reactionreceptacles to prescribed temperature conditions and including signaldetectors for detecting signals emitted by the contents of the reactionreceptacles during an incubation process.

FIG. 4 is a bottom plan view of a receptacle carrier carousel of theincubator.

FIG. 5 is a perspective view of assembled components of a receptaclecarrier carousel of the incubator and a circulating fan for generatingairflow within the incubator.

FIG. 6 is a perspective view of a bottom wall of the incubator housing,a portion of the receptacle carrier, and a receptacle carrier driveassembly.

FIG. 7 is a perspective view of a receptacle divider of the receptaclecarrier.

FIG. 8 is a perspective view of the receptacle divider from an oppositeside of the divider.

FIG. 9 is a partial perspective view of components of the receptaclecarrier of the incubator including a receptacle presence sensor fordetecting the presence of reaction receptacles on the receptaclecarrier.

FIG. 10 is a partial perspective view of a portion of the incubatorincluding the incubator floor, signal detectors disposed beneath theincubator floor, and reaction receptacles disposed in signal detectingpositions with respect to the signal detectors.

FIG. 11 is a perspective view of a signal detector for use inconjunction with the present invention.

FIG. 12 is a bottom plan view of the signal detector.

FIG. 13 is a side cross-sectional view of the signal detector takenalong the line 13-13 in FIG. 12.

FIG. 14 is an exploded perspective view of the signal detector.

FIG. 15 is a partial top plan view of a lower disk of the receptaclecarrier carousel of the incubator, showing alignment of a multiplereceptacle device carried on the receptacle carrier with a signaldetector positioned below the receptacle carrier and the relativeorientations of fluorescent standards mounted on the lower disk relativeto the signal detector.

FIG. 16 is a partial side cross sectional view (along ling I-I in FIG.15) showing a signal detector and a receptacle carried on the receptaclecarrier in a detection zone with respect to the signal detector.

FIG. 17 is a partial side cross sectional view (along ling I-I in FIG.15) showing the signal detector, the receptacle moved out of thedetection zone with respect to the signal detector, and a fluorescentstandard moved into optical communication with the signal detector butnot in the detection zone with respect to the signal detector.

FIG. 18 is a flow chart showing a self-check procedure embodying aspectsof the present invention for a fluorometer or other optical signaldetector.

FIG. 19 is a graph showing excitation spectra of preferred amplificationdetection dyes.

FIG. 20 is a graph showing emission spectra of preferred amplificationdetection dyes.

FIG. 21 is a graph showing excitation and emission fluorescence spectrafor FAM, HEX, and ROX dyes.

FIG. 22 is a block diagram schematically illustrating excitation anddetection architecture.

FIG. 23 is a block diagram schematically illustrating an arrangement ofdetection circuitry.

FIG. 24 is a block diagram schematically illustrating an arrangement ofexcitation circuitry.

FIG. 25 is a circuit diagram illustrating a fluorometer excitationcircuit.

FIGS. 26A and 26B are two parts of a circuit diagram illustrating afluorometer detection circuit.

FIG. 27 is a flow chart showing the protocols of an exemplary real-timeamplification assay.

FIG. 28 is a flow chart showing an analyte quantification process.

FIG. 29 is a time plot of real-time fluorometer data.

FIG. 30 is a plot showing a method for fitting a curve to real-timefluorometer data and using the fit to determine a threshold time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Multiple Receptacle Devices

Referring to FIG. 1, a reaction receptacle in the form of a multiplereceptacle device (“MRD”) 160 comprises a plurality of individualreceptacle vessels, or reaction tubes, 162, preferably five. Thereceptacle vessels 162, preferably in the form of cylindrical tubes withopen top ends and closed bottom ends, are connected to one another by aconnecting rib structure 164 which defines a downwardly facing shoulderextending longitudinally along either side of the MRD 160.

The MRD 160 is preferably formed from injection molded polypropylene,such as those sold by Montell Polyolefins, of Wilmington, Del., productnumber PD701NW or Huntsman, product number P5M6K-048. In an alternativeembodiment, the receptacle vessels 162 of the MRD are releasably fixedwith respect to each other by means such as, for example, a sample tuberack.

An arcuate shield structure 169 is provided at one end of the MRD 160.An MRD manipulating structure 166 extends from the shield structure 169.The manipulating structure is adapted to be engaged by a transportmechanism for moving the MRD 160 between different components of adiagnostic analyzer. An exemplary transport mechanism that is compatiblewith the MRD 160 is described in U.S. Pat. No. 6,335,166. The MRDmanipulating structure 166 comprises a laterally extending plate 168extending from shield structure 169 with a vertically extending piece167 on the opposite end of the plate 168. A gusset wall 165 extendsdownwardly from lateral plate 168 between shield structure 169 andvertical piece 167. Vertical piece 167 comprises a convex face 171facing the shield structure 169.

As shown in FIG. 2, the shield structure 169 and vertical piece 167 havemutually facing convex surfaces. The MRD 160 may be engaged by atransport mechanism and other components, by moving an engaging memberlaterally (in the direction “A”) into the space between the shieldstructure 169 and the vertical piece 167. The convex surfaces of theshield structure 169 and vertical piece 167 provide for wider points ofentry for an engaging member undergoing a lateral relative motion intothe space.

A label-receiving structure 174 having a flat label-receiving surface175 is provided on an end of the MRD 160 opposite the shield structure169 and MRD manipulating structure 166. Human and/or machine-readablelabels, such as scannable bar codes, can be placed on the surface 175 toprovide identifying and instructional information on the MRD 160.

Further details regarding the MRD 160 may be found in U.S. Pat. No.6,086,827.

Nucleic Acid Diagnostic Assays

Aspects of the present invention involve apparatus and procedures thatcan be used in conjunction with nucleic acid diagnostic assays,including “real-time” amplification assays and “end-point” amplificationassays.

Real-time amplification assays can be used to determine the presence andamount of a target nucleic acid in a sample which, by way of example, isderived from a pathogenic organism or virus. By determining the quantityof a target nucleic acid in a sample, a practitioner can approximate theamount or load of the organism or virus in the sample. In oneapplication, a real-time amplification assay may be used to screen bloodor blood products intended for transfusion for bloodborne pathogens,such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV).In another application, a real-time assay may be used to monitor theefficacy of a therapeutic regimen in a patient infected with apathogenic organism or virus, or that is afflicted with a diseasecharacterized by aberrant or mutant gene expression. Real-timeamplification assays may also be used for diagnostic purposes, as wellas in gene expression determinations.

In addition to implementation of the invention in conjunction withreal-time amplification assays, the invention may also be implemented inconjunction with end point amplification assays. In end-pointamplification assays, the presence of amplification products containingthe target sequence or its complement is determined at the conclusion ofan amplification procedure. The determination may occur in a detectionstation that may be located externally to the incubator in which theamplification reactions occur. In contrast, in “real-time” amplificationassays, the amount of amplification products containing the targetsequence or its complement is determined during an amplificationprocedure. In the real-time amplification assay, the concentration of atarget nucleic acid can be determined using data acquired by makingperiodic measurements of signals that are functions of the amount ofamplification product in the sample containing the target sequence, orits complement, and calculating the rate at which the target sequence isbeing amplified from the acquired data.

In an exemplary real-time amplification assay, the interacting labelsinclude a fluorescent moiety, or other emission moiety, and a quenchermoiety, such as, for example, 4-(4-dimethylaminophenylazo) benzoic acid(DABCYL). The fluorescent moiety emits light energy (i.e., fluoresces)at a specific emission wavelength when excited by light energy at anappropriate excitation wavelength. When the fluorescent moiety and thequencher moiety are held in close proximity, light energy emitted by thefluorescent moiety is absorbed by the quencher moiety. But when a probehybridizes to nucleic acid present in the sample, the fluorescent andquencher moieties are separated from each other and light energy emittedby the fluorescent moiety can be detected. Fluorescent moieties whichare excited and emit at different and distinguishable wavelengths can becombined with different probes. The different probes can be added to asample, and the presence and amount of target nucleic acids associatedwith each probe can be determined by alternately exposing the sample tolight energy at different excitation wavelengths and measuring the lightemission from the sample at the different wavelengths corresponding tothe different fluorescent moieties.

Where an amplification procedure is used to increase the amount oftarget sequence, or its complement, present in a sample before detectioncan occur, it is desirable to include a “control” to ensure thatamplification has taken place and, thereby, to avoid false negatives.Such a control can be a known nucleic acid sequence that is unrelated tothe sequence(s) of interest. A probe (i.e., a control probe) havingspecificity for the control sequence and having a unique fluorescent dye(i.e., the control dye) and quencher combination is added to the sample,along with one or more amplification reagents needed to amplify thecontrol sequence, as well as the target sequence(s). After exposing thesample to appropriate amplification conditions, the sample isalternately exposed to light energy at different excitation wavelengths(including the excitation wavelength for the control dye) and emissionlight is detected. Detection of emission light of a wavelengthcorresponding to the control dye confirms that the amplification wassuccessful (i.e., the control sequence was indeed amplified), and thus,any failure to detect emission light corresponding to the probe(s) ofthe target sequence(s) is not likely due to a failed amplification.Conversely, failure to detect emission light from the control dye may beindicative of a failed amplification, thus rendering any results fromthat assay suspect. Alternatively, failure to detect emission light maybe due to failure or deteriorated mechanical and/or electricalperformance of an instrument (described below) for detecting theemission light. The present invention concerns methods and apparatus fordetecting such failure or deteriorated performance. In the presentcontext, “performance” means the reliability of the operation of theinstrument such that output of the instrument may be relied upon inderiving an assay or test result. Instrument failure or deterioratedperformance may be detected, in accordance with principles of theinvention, by objectively measurable characteristics of the output ofthe instrument that deviate from an output that would be normallyexpected under similar operation conditions if the instrument wereoperating properly. Examples of such objectively measurablecharacteristics that may be indicative of instrument failure ordeteriorated performance may include an unexpected decrease in theintensity of the instrument output or a spike in the instrument output.

Systems and methods for performing real-time amplification assays aredescribed in Macioszek et al., “Methods for Performing Multi-Formattedassays,” U.S. Pat. No. 7,897,337. Systems and methods for end-pointdetection are described in Ammann, et al., “Automated Process ForIsolating and Amplifying a Target Nucleic Acid Sequence” U.S. Pat. No.6,335,166.

In accordance with aspects of the present invention, amplificationassays are performed in an incubator, such as incubator 200, features ofwhich are shown in FIGS. 3-10. Incubator 200 is a rotary incubator inthe sense that MRDs 160 are carried on a rotary carrier structure (e.g.,a carousel) within a controlled temperature housing. Incubator 200includes signal detectors, or signal detector blocks, 400 attachedthereto for detecting, in a real-time manner, the amplificationoccurring within the reaction tubes 162 of an MRD 160 carried in theincubator, for example, by measuring the fluorescence emitted by a dyeor dyes within each reaction tube 162 of the MRD 160 when the MRD 160 isilluminated with an excitation light corresponding to each dye. Theincubator 200 can be integrated into an automated diagnostic analyzer(not shown) that may include one or more receptacle transport mechanismsfor placing MRDs 160 into the incubator 200 and removing MRDs 160 fromthe incubator 200.

Features of an incubator 200, adapted for use in conjunction with thepresent invention, are shown in FIGS. 3-10. FIG. 3 shows an explodedperspective view of the incubator 200. The incubator 200 includes ahousing that comprises an outer wall 202, a bottom wall 206, and a topwall (not shown), all of which are covered by a thermal insulatingshroud, or hood, 212, which is shown lifted off the remainder of theincubator. The side, bottom and top walls are preferably formed ofaluminum, and the insulating hood is preferably made from a suitableinsulating material, such as polyurethane foam. A receptacle carrier242, preferably in the form of a carousel rotatably mounted within thehousing, is configured for carrying a plurality of reaction receptacles.Receptacles, such as MRDs 160, can be inserted into the receptaclecarrier 242 and removed from the receptacle carrier 242 through areceptacle opening 204 formed in the sidewall 202. Receptacle opening204 is covered by the sliding door 216 of a door assembly 214 (describedin more detail below).

One or more signal detectors 400 are disposed beneath the bottom wall206 of the incubator housing and are configured for detecting signalsemitted by the contents of MRDs 160 carried on the receptacle carrier242 within the incubator 200. The signal detectors 400, which maycomprise fluorometers for detecting fluorescent signals, are describedin further detail below.

Heat may be generated within the incubator 200 by any suitable means. Inone embodiment, resistive heating elements are disposed on the sidewall202 of the incubator housing. Other suitable heating elements mayinclude, for example, Peltier' thermoelectric heating elements. Theheating elements may be under microprocessor control for maintaining aconstant, desired temperature, and the incubator 200 may further includeone or more temperature-sensing elements for providing temperature levelsignals to the microprocessor controller.

A circulating fan 226 may be positioned within the incubator housing,for example, atop the receptacle carrier 242. In one embodiment, fan 226is an axial fan, as shown, configured for generating airflow through thereceptacle carrier 242 and within the incubator 200.

Further details concerning the construction of the receptacle carrier242 are shown in FIGS. 4 and 5. FIG. 4 is a bottom plan view of thereceptacle carrier 242 with a plurality of MRDs 160 carried thereon.FIG. 5 is a perspective view of a portion of the receptacle carrier 242and showing the fan 226 mounted atop the carrier 242.

Carrier 242 comprises an upper disk 244 and an identical lower disk 256.As shown in FIGS. 4 and 5, the lower disk includes an inner ring 258, anouter ring 260, and an intermediate ring 262 disposed concentricallybetween the inner ring 258 and outer ring 260. Inner radial spokes 266extend between the inner ring 248 and the intermediate ring 262. Outerspokes 264 extend between the intermediate ring 262 and the outer ring260 and are, in this embodiment, in a non-radial orientation, meaningthat each spoke is configured obliquely with respect to a true-radialorientation relative to the center of the intermediate ring 262 andouter ring 260. Optical reference standards 250 are provided on selectedouter spokes 264 of the lower disk 256 of the receptacle carrier 242.The purpose of these optical reference standards 250 will be describedbelow.

The upper disk 244 has a similar construction, but only outer ring 248and outer spokes 252 are visible in FIG. 5. The upper disk 244 furtherincludes an inner ring, an intermediate ring, and inner spokes, all ofwhich are obstructed from view by the fan 226 in FIG. 5.

The upper disk 244 and the lower disk 256 are secured relative to oneanother in a parallel, spaced-apart orientation by a plurality of spacerposts 268 disposed at angular intervals around the perimeters of theupper disk 244 and lower disk 256. Each spacer post 268 may be securedin place by means of a suitable fastener, such as a screw, extendingthrough a hole in the upper disk 244 or lower disk 256 and into anopening (e.g. a threaded opening) formed in each end of each of thespacer posts 268.

The receptacle carrier 242 further includes a plurality of receptacledividers 274 extending between each of the outer spokes 264 of the lowerdisk 256 and corresponding outer spokes 252 of the upper disk 244. Thespaces between adjacently disposed receptacle dividers 274 definereceptacle stations 240, each configured to receive a single MRD 160. Asshown in FIG. 4, which is a bottom plan view of a receptacle carriercarousel of the incubator, each MRD 160 is carried in a generallyvertical orientation with the lower ends of each receptacle vessel 162exposed at the bottom of the receptacle carrier 242 and with thereceptacle manipulating structure 166 of each MRD 160 extending radiallybeyond the outer perimeter of the receptacle carrier 242.

Details of the receptacle dividers are shown in FIGS. 6-8. As noted,each receptacle divider 274 is attached to one of the outer spokes 264of the lower disk 256, as shown in FIG. 6. The receptacle divider 274includes a divider wall 276 that is oriented generally vertically whenthe divider 274 is installed between the upper disk 244 and lower disk256. The divider wall 276 includes lower positioning posts 278configured to be inserted into mating openings formed in the lower disk256 (not shown) and upper positioning posts 280 configured to beinserted into mating openings (not shown) formed in the upper disk 244.In an embodiment of the invention, the incubator 200 holds eighteen MRDs160 at a time, each spaced at 20° increments around the carousel.

A drive assembly 300 of the receptacle carrier 242 includes a motor 302mounted on a motor mount portion 208 of the bottom wall 206 of theincubator housing, guide wheels 304 and 306, and a drive belt 308. Drivebelt 308 is secured around a drive shaft (not shown) of the motor 302,around the guide wheels 304 and 306, and further over the belt drivesupports 298 of the plurality of dividers 274 mounted between the upperdisk 244 and lower disk 256. As noted, each drive belt support 298 mayinclude a vertical rib 299 for engaging teeth (not shown) of the drivebelt 308. As shown in FIG. 6, which shows a perspective view of a bottomwall of the incubator housing, a portion of the receptacle carrier, anda receptacle carrier drive assembly, the bottom wall 206 of theincubator housing includes a plurality of elongated openings 210,preferably formed at equal angular intervals about a point correspondingto the axis of rotation of the receptacle carrier 242. The openings 210are oriented at the same angle at which each MRD 160 will be orientedwhen carried on the receptacle carrier 242, and each opening 210 isconfigured to receive an upper end of a signal detector 400 extendinginto the incubator 200 for detecting signals emitted by the contents ofthe MRDs 160 during the incubation process. Motor 302 is preferably astepper motor under microprocessor control to enable precise control ofrotation of the receptacle carrier 242. A “home” position sensor (notshown) indicates when the receptacle carrier 242 is in a specifiedrotational position, and the motor 302 is provided with an encoder.Accordingly, movement of the receptacle carrier 242 can be controlled,e.g., by a microprocessor receiving signals from the home sensor and anencoder coupled to motor 302 to control and monitor the angular movementand positioning of the carrier 242, to sequentially place each MRD 160on the receptacle carrier 242 into a signal detection position above theopenings 210.

As shown in FIG. 9, which shows a partial perspective view of componentsof the receptacle carrier of the incubator, the receptacle carrier 242further includes a center post 270 extending between the inner ring 258of the lower disk 256 and the inner ring of the upper disk 244 (notshown in FIG. 9). A receptacle presence sensor 272 is mounted to thecenter post 270 and is configured to detect the presence of an MRD 160inserted into a receptacle station 240 of the receptacle carrier 242.Microprocessor control, which controls and monitors the angular positionof the receptacle carrier 242, also monitors the location of eachspecific MRD 160, which may be identified by, e.g., a label, such as amachine-readable bar-code or an RFID tag. That is, when an MRD 160,identified via its label or other means, is moved into the incubator200, the angular position of the receptacle station 240 into which thatMRD 160 is inserted is determined and tracked to monitor the position ofthat MRD 160 at all times while the MRD is inside the incubator 200.

With reference to FIGS. 9 and 10-14, the signal detectors 400 are partof a system that measures, for example, the concentration of unquenchedfluorescent dye molecules within the contents of receptacle vessels 162of MRDs 160 carried on the receptacle carrier 242. The assay performedwithin each receptacle vessel 162 of each MRD 160 may be designed suchthat the fluorescent signal increases as the concentration of target isincreased by amplification. The signal detector 400 (e.g., afluorometer) is used to monitor the amplification process by monitoringthe emergence of the fluorescent signal.

An exemplary embodiment of the incubator 200 may include between threeand six signal detectors 400, where each detector is designed to measurea particular fluorescent dye (i.e., color). Each signal detector 400houses, for example, five individual detectors. The five individualdetectors (also referred to herein as “channels”) are spaced relative toeach other with the same spacing as that of the receptacle vessels 162of each MRD 160. The signal detector 400 may be provided with additionalor fewer individual detectors, but the number of detectors generallycorresponds to the number of receptacle vessels 162 in each MRD 160. Thesignal detectors 400 are mounted to the amplification incubator 200 withsuch an orientation that each of them can detect signal emitted by thecontents of each receptacle vessel 162 of an MRD 160 when the receptaclecarrier 242 stops at preset angular increments corresponding to theangular positions of the signal detectors 400. Therefore, each MRD 160can be scanned by each signal detector 400 once per revolution of thecarrier 242.

As shown in FIG. 10, which is a partial perspective view of a portion ofthe incubator, in one embodiment, six signal detectors 400 areconstructed and arranged to detect signals emitted by the contents ofeach of the five receptacle vessels 162 of six different MRDs 160carried within the housing of the incubator 200. That is, each signaldetector 400 is configured to detect a signal emitted by each of thefive receptacle vessels 162 of an MRD 160 operatively positioned withrespect to the signal detector 400 by the carrier 242. The signaldetectors 400 may be of substantially identical constructions, but eachmay be adapted to detect a signal characteristic of a differentmeasureable or detectable value. For example, each signal detector 400may be configured to detect fluorescence of a different wavelength(i.e., color), and thus each may be configured, or tuned, to detect adifferent fluorescent dye within the contents of the receptacle vessel162. Each signal detector 400 may also be configured to emit light at apredefined wavelength or within a range of wavelengths. The wavelengthof the emitted light from the signal detector 400 frequently correspondsto an excitation wavelength window of a fluorescent dye within thecontents of the receptacle vessel 162.

The motor 302, which drives the receptacle carrier 242, is under thecontrol of a microprocessor which may receive signals from a home sensorcoupled to the carrier 242, a timer, and an encoder coupled to the motor302 for controlling movement and angular positioning of the carrier 242.The carrier 242 is controlled to (a) move MRD(s) 160 into operative,sensing positions with respect to the signal detector(s) 400, (b) pausefor a sufficient period of time to permit the signal detector(s) to takeand process a signal reading from the MRD operatively positioned withrespect to it, and (c) index the carrier 242 to position the next MRD(s)160 into operative position(s) with respect to the signal detector(s)400.

Details of a signal detector 400 for use in conjunction with the presentinvention are shown in FIGS. 11-14. As shown in FIG. 11, which is aperspective view of a signal detector, the detector 400 includes ahousing that comprises a detector housing 418 and an excitation housing402, both connected at a right angle with respect to each other to alens and filter, or optics, housing 434. An interface cap 456 isattached to the optics housing 438. Each of the housing components 402,418 and 434 may be made from, for example, machined aluminum and securedto one another by suitable fasteners, such as screws, and is preferablyanodized. The interface cap 456 is preferably machined fromnon-thermally conductive material, such as Delrin®, so as to minimizethermal conduction between the incubator 200 and the detector 400. Anexcitation printed circuit board (“PCB”) 406 is connected to an end ofthe excitation housing 402, and a detector PCB 422 is connected to anend of the detector housing 418. Excitation and detector circuitrydisposed on the excitation PCB 406 and the detector PCB 422,respectively, are described below. A flexible cable 454 connects theexcitation PCB 406 with the detector PCB 422.

The interface cap 456 includes a rim flange 460 surrounding theperiphery of the cap 456 and a dome portion 458 projecting above the rimflange 460. As shown, for example, in FIG. 10, the dome 458 of theinterface cap 456 extends into the detector opening 210 formed in thebottom wall 206 of the incubator 200, and the rim flange 460 abuts thebottom portion of the bottom wall 206 surrounding the detector opening210 so as to provide a light-tight seal between the interface cap 456and the bottom wall 206. A gasket material may be provided between therim flange 460 and the bottom wall 206 to further enhance thelight-tight seal. Five detection openings 462 are provided in theinterface cap 456.

As shown in FIGS. 13 and 14, which show a side cross-sectional view andan exploded perspective view, respectively, of the signal detector, theexcitation housing 402 includes five excitation channels 404. Anexcitation light source 405, such as a light-emitting diode (“LED”)coupled to the excitation PCB 406 is located at the end of eachexcitation channel 404. Similarly, the detector housing 418 includesfive emission channels 420, and a detector element, or optical signaldetector, 423, such as a photodiode, is provided in each emissionchannel 420 and is coupled to the detector PCB 422. A standoff 464 ismounted between the excitation housing 402 and the detector PCB 422 at adistance from the detector housing 418 to provide additional stabilityfor the detector PCB 422.

Within each individual channel of each detector 400 there are twooptical paths defined by excitation optics and emission optics disposed,at least partially, within the excitation and emission channels,respectively. As described in more detail below, the excitation opticalpath begins with an LED as the light source, which light is collimatedby an excitation lens and then filtered through an excitation filter.The filtered light passes upward through a beam splitter and is focusedonto a receptacle vessel 162 by objective lenses between the receptaclevessel 162 and the beam splitter. The emission optical path originatesfrom the light emitted by the contents of the receptacle vessel 162,which is collimated by the objective lenses as the light passes towardthe beam splitter and is reflected by the beam splitter toward theemission channel. Within the emission channel, after being filteredthrough an emission filter, the light is focused by an emission lensonto the detector element 423, such as a photodetector.

The various optical elements of the detector 400 are located in theoptics housing 434. For each excitation channel 404 of the excitationhousing 402, the optics housing 434 contains excitation optics 408, foreach emission channel 420 of the detector housing 418, the opticshousing 434 contains emission optics 424, and for each detector opening462 of the interface cap 456, the optics housing 434 containsinput/output optics 444. The excitation optics 408, emission optics 424,and input/output optics 444 are disposed within optics channels 436formed within the optics housing 434.

The excitation optics comprises an optical focus and filter assembly andinclude an excitation lens 412, a lens holder 414, and an excitationfilter 416. An O-ring 410 provides a light-tight seal between theexcitation housing 402 and the optics housing 434. The excitation filter416 is selected so as to pass excitation light from the light source 405within the excitation channel 404 having a desired excitationcharacteristic (e.g., wavelength).

The emission optics include an emission lens 428, a lens holder 430 andan emission filter 432. An O-ring 426 provides a light-tight sealbetween the detector housing 418 and the optics housing 434. Theemission filter 432 is selected so as to transmit only that portion of asignal emitted by the contents of a reaction receptacle to the detector423 within the emission channel 420 having a desired signalcharacteristic (e.g., wavelength).

The input/output optics 444 include a first objective lens 450 and asecond objective lens 448 with a spacer ring 446 disposed therebetween.An O-ring 452 provides a light-tight seal between the interface cap 456and the optics housing 434.

The detector 400 further includes dichroic beam-splitters comprisingdichroic beam-splitter elements 440 held within a beam-splitter frame442 which is inserted into a beam-splitter opening 438 of the opticshousing 434. A beam-splitter 440 is provided for each excitation channel404 and corresponding emission channel 420. The beam-splitter 440 isselected so as to pass excitation light having a prescribed excitationwavelength in a straight optic path from the excitation channel 404 andto deflect emission light from the contents of the receptacle 162 havinga prescribed detection wavelength toward the detection channel 420.

In one embodiment, the signal detector comprises a fluorometerconfigured to excite a fluorescent dye of a specific wavelength (i.e.,color), by directing an optical excitation signal of a specified,associated excitation wavelength at a receptacle containing a samplewith which the fluorescent dye is mixed, and to detect an emissionsignal having a wavelength corresponding to the wavelength, or color, ofthe specific dye. Different fluorescent dyes are excited at differentwavelengths. In one multiplex application of the present invention,suitable dyes include the rhodamine dyes tetramethyl-6-rhodamine(“TAMRA”) and tetrapropano-6-carboxyrhodamine (“ROX”) and thefluorescein dyes 6-carboxyfluorescein (“FAM”) and, each in combinationwith a DABCYL quencher. Other suitable dyes include5′-hexachlorofluorescein phosphoramidite (“HEX”), and2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (“JOE”). Thenormalized excitation spectra of FAM, JOE, TAMRA, and ROX dyes are shownin FIG. 19. FIG. 20 shows the normalized emission spectra of the FAM,JOE, TAMRA, and ROX dyes. Because the preferred dyes are excited atdifferent wavelengths, each signal detector 400 is preferably tailoredto emit an excitation light at or near the desired excitation wavelength(i.e., color) for the particular dye that the fluorometer is intended todetect. Accordingly, component selection for the detector/fluorometerwill, in many instances, be governed by the particular dye for which thesignal detector 400 is intended. For example, with respect to the lightsource 405, the particular LED selection will depend on the dye forwhich the fluorometer is intended. As shown in FIG. 21, which showsnormalized excitation and emission fluorescence versus wavelength forFAM, HEX, and ROX dyes, the HEX excitation wavelength band partiallyoverlaps with FAM emission wavelength band, and the ROX excitationwavelength band partially overlaps with HEX emission wavelength band.See also Table 1 below.

The detectors 400 are identical in design and components, with theexception of components that are dye-specific. The components that aredye-specific include the light source 405, the excitation filter 416,the emission filter 432, and the beam splitter 440.

The following table provides specifications for a selection of filtersfor different types of dyes:

Filter Specifications

TABLE 1 Center Wavelength Bandwidth Dimensions Description (nm) (nm)(mm) Thickness FAM Excite Filter 460 60 8.9 × 8.9 2 square FAM Emission525 30 8.9 × 8.9 2 Filter square FAM Short Wave 10 × 14.8 1.05 PassDichroic rectangular HEX Excite Filter 535 22 8.9 × 8.9 2 square HEXEmission 567 15 8.9 × 8.9 2 Filter square HEX Short Wave 10 × 14.8 1.05Pass Dichroic rectangular ROX Excite Filter 585 29 8.9 × 8.9 2 squareROX Emission 632 22 8.9 × 8.9 2 Filter square ROX Short Wave 10 × 14.81.05 Pass Dichroic rectangular

The following table provides specifications for a selection of lensesfor different types of dyes:

Lens and O-Ring Specifications

TABLE 2 Dye = FAM, HEX, ROX Part No. Description Vendor NT47-475Emission Lens Edmund or Ross NT47-477 Excitation Lens Edmund or RossNT47-476 Objective Lens Edmund or Ross 94115K478 O-ring McMaster

Table 3 shows certain preferred characteristics of exemplary blue,green, and amber LED:

LED Specifications

TABLE 3 Characteristic Blue Green Amber Chip Size 24 mil 11 mil 25 milDominant Wavelength 462 nm 533 nm 590 nm Radiant Flux 4 mW 2 mW 1.2 mWMax DC forward 200 mA 50 mA 150 mA current

Note that in the illustrated embodiment, the beam splitter 440 passesthe excitation light and reflects the emission light. Since theexcitation channel is longer than the emission channel, this arrangementprovides a narrow profile for the housing of the signal detector 400,thereby maximizing the number of detectors 400 that can be positioned atangular intervals beneath the incubator 200, as shown in FIG. 10.Spatial limitations and preferences may be accounted for in designingthe excitation and emission channels, which can be interchanged from theformat depicted in FIG. 10. In such an embodiment a beam splitter thatreflects the excitation light and passes the emission light could beused.

The data acquisition system and process for acquiring, storing, andprocessing signal data emitted by the contents of the MRDs 160 can bedescribed at a high level with reference to FIG. 22. In general, thesystem and process include three components: excitation 406, detection422, and control 506. The Excitation branch, or circuitry, 406 generatesa power signal to control the light source (e.g., an LED) to generate anexcitation light signal. The detection branch, or circuitry, 422includes a light detector (e.g., a photodiode) that converts photons oflight that impinge on the detector to a current. The control branch, orcontroller, 506 drives and controls the excitation circuitry 406 andprocesses the emission data generated by the detection circuitry 422.

FIG. 23 depicts a logical block diagram of an arrangement of thedetection circuitry 422. The detection circuitry on the detector PCB 422can include detector circuits 502 a-502 e, which are configured todetect fluorescent light and to convert the detected light to a voltagesignal that can be processed by the controller 506. The output from thedetector circuits 502 a-502 e can be connected to controller 506 eitherdirectly or through a multiplexer 504, as is shown in FIG. 23.

FIG. 24 depicts a logical block diagram of an arrangement of theexcitation circuitry. As shown in FIG. 24, which is a block diagramschematically illustrating an arrangement of excitation circuitry,excitation circuitry can include the controller 506 and a digital toanalog converter (DAC) 510. The excitation circuitry on the excitationPCB 406 includes excitation circuits 512 a-512 e for driving eachexcitation source 405 of each excitation channel 404. The excitationcircuits 512 a-512 e are driven by a digital to analog converter (DAC)controlled current source. The current source is a voltage to currentamplifier that controls the current flowing through the excitationsource 405.

A monitor 516 can be connected to excitation circuits 512 a-512 e tofacilitate process control of the excitation voltage. Checking thevoltage across the LED and the current through the LED give a goodindication if the LED is functioning correctly. This is a diagnosticcapability that can be used in a variety of ways. For example, the LEDcould be checked at power-on, during a self test, so when thefluorometer powers up it could put a known current through the LED, andif the forward voltage of the LED is in an expected range, then thesystem would pass the self test. These values could also be checkedduring an assay to monitor correct functioning of the LED.

According to embodiments of the invention, each of the LEDs(corresponding to excitation source 405) in circuits 512 a-512 e can bedriven by a digital to analog converter controlled current source, asshown in FIG. 25, which is a circuit diagram illustrating a fluorometerexcitation circuit. The current source can be a voltage to currentamplifier that controls the current flowing through the LED(corresponding to excitation source 405).

In addition to performing the function of driving a computer controlledcurrent waveform through the LED, the current source shown in FIG. 25allows for process control based on LED current and voltage. The outputof the circuit formed by U3B is a monitor of the voltage across the LEDand can be digitized by monitor 516 using an A/D converter. Similarlythe output of R22 (the side away from transistor Q3) can be used tomonitor the current passing through the LED and similarly digitized byan A/D converter located in monitor 516. The current through the LED ismonitored for diagnostic purposes, as described above.

The detector circuits 502 a-502 e could be configured as shown in FIGS.26A and 26B. Each detector circuit 502 includes a pre-amplifier circuit,which includes U11, and the amplifier formed by pins 5-7 of U10B. Thepre-amplifier circuit receives current from the photodiode D 473(corresponding to detector element 423) and converts it to an amplifiedvoltage. As shown, the amplifier that includes pins 1-3 of U10A providesa bias current to compensate for the electrical current out ofphotodiode D caused by un-modulated ambient light incident on thephotodiode D.

Amplifiers U11 and U10B form the first two stages of amplification ofthe current signal (corresponding to the emission signal) from thephotodiode D (423). C54, C44, and C58 provide power supplybypassing/filtering to the amplifiers. C12, D12, R55, R57, and R61 forma filtered power supply that biases the anode of the photodiode D.Feedback resistors R31 and R32 convert electrical current from thephotodiode D into a voltage while C48 provides filtering for higherfrequency signals. The voltage divider formed by R43 and R45 provides avoltage gain of 10 in the next pre-amplification state while capacitorC56 provides additional low pass filtering.

The detector circuits 502 a-502 e are configured to use a level shifterformed by U7A. Though not wishing to be bound by theory, the purpose ofthe level shifter is to move the zero level of the pre-amp up to themiddle range of a unipolar analog to digital converter. This allows theuse of A/D converters employed by certain microcontrollers so that anadditional A/D converter is not required.

During operation, while multiple receptacles (e.g., MRDs 160) are beingprocessed within the incubator 200 and one or more signal detectors 400are measuring the intensity of signal emissions from the receptaclescarried on the receptacle carrier 242, it is desirable to periodicallyself-check the signal detectors to detect any failure or deterioratedperformance. Such a failure or performance deterioration can affect theaccuracy of test results, which hinge on measurement of opticalemissions from the sample tubes. In general, such self-checking isperformed in accordance with aspects of the present invention by movingan optical reference standard 250 (e.g., a fluorescent referencestandard) into optical communication with each channel of each signaldetector 400 (or in the case of a non-stationary detector, moving thedetector into optical communication with an optical reference standard),measuring the optical emission intensity from the reference standard,and comparing the measured intensity to an expected intensity. Adifference between the measured and expected intensities that exceeds athreshold may be indicative of failure or deteriorated performance ofthe signal detector. In the case of a stationary detector, eachreference standard may be carried on a support with respect to thestationary signal detectors 400 to periodically place the referencestandard in optical communication with a channel of the signal detector.“Optical communication” in the present context refers to positioning theoptical reference standard in a position with respect to a channel ofthe signal detector, or positioning a channel of the signal detector ina position with respect to the optical reference standard, such that thesignal detector is able to detect an optical emission from the standard.In the illustrated embodiment, as shown in FIGS. 4, 5, 6, and 9, opticalreference standards 250 are provided on selected outer spokes 264 of thelower disk 256 of the receptacle carrier 242. These standards arepositioned and oriented in this embodiment such that as the carrier 242rotates within the incubator 200, one or more reference standards 250can be moved into a position with respect to a channel of a signaldetector 400 that will enable the signal detector 400 to measure theoptical emission from the standard 250. In one embodiment, the opticalreference standard is a fluorescent reference standard made from afluorescent material that will provide an elevated relative fluorescentsignal when the standard is placed in optical communication with achannel of a signal detector 400. That is, the fluorescent standard willgenerate a sufficiently high fluorescent emission signal that failure ordeteriorated performance of a signal detector will manifest itself in adetectable change in the measured fluorescent emission intensity fromthe standard.

In the illustrated embodiment, each fluorescent reference standard 250comprises a single fluorescent plastic disk secured onto an outersurface of an outer spoke 264 of the lower disk 256 as a self-checktarget for each of the five channels of the signal detector 400. Eachgroup of five reference standards 250 (corresponding to the number ofchannels of each signal detector 400) on one outer spoke 264 comprises areference standard set, and the number of reference standard setspreferably corresponds to the number of signal detectors 400 positionedbeneath the incubator 200 so that all of the signal detectors can beself-checked simultaneously. That is, in one embodiment, each referencestandard set corresponds to, or is associated with, one signal detector.In other embodiments, reference standard sets are positioned relative totheir corresponding signal detector so that one or more, but less thanall, signal detectors can be self-checked while one or more other signaldetectors are measuring signal emissions from samples.

By integrating the reference standard into a support structure, such asthe receptacle carrier 242, within the incubator 200, the signaldetector(s) 400 can be self-checked during operation of the incubator200, including during the real-time monitoring of amplificationreactions within the incubator. Thus, the self-check procedure can beperformed within the closed system of the incubator without requiringthat normal operation of the incubator be interrupted to permit thesignal detector(s) to be checked for proper operation.

Suitable materials for the fluorescent standards 250 frequently compriseconsumer-grade fluorescent plastic sheets from which disks (of, e.g.,3.5 mm diameter) can be cut or stamped and secured to the outer spokes264. The fluorescent plastic discs may be secured to the spoke by asuitable adhesive and/or pressed into a friction fit within a, forexample, circular hole formed in the spoke 264. Forming holes in theouter spokes 264 for receiving the reference standards 250 whilemachining the lower disk 256 of the receptacle carrier 242 will simplifythe later process of securing the reference standards 250 to the disk256 and will ensure that the references standards 250 are properlylocated on the disk 256. Suitable plastic sheets include fluorescentplastic sheet available from McMaster-Car (Part No. 85635K412, colorAmber) manufactured by Reynolds Polymer and Acrycast Cell Cast AcrylicSheet (Part No. 2422, color Amber). Preliminary tests indicate that theMcMaster-Car Amber plastic is a preferable reference standard materialfor self-checking fluorometers configured to detect ROX dyes, and theAcrycast Cell Cast Acrylic amber is a preferable reference standardmaterial for self-checking fluorometers configured to detect FAM and HEXdyes. Other colors may be suitable for reference standards, such asblue, pink, and green.

The colors, or other emission characteristics, of reference standards ofdifferent reference standard sets may all be the same, or differentcolors may be used in different reference standard sets forself-checking fluorometers, or other signal detectors, configured todetect different colors. Furthermore, all the reference standards ineach reference standard set may be the same color, or different coloredreference standards may be used in a reference standard set if thedifferent channels of the signal detector are configured to detectdifferent colors.

One of skill in the art may appreciate that a variety of materials existthat have that have overt fluorescent characteristics, producing strongfluorescence signals when excited with appropriate light wavelengths(e.g., the fluorescent plastics described above). Many more materialshave residual fluorescent characteristics, which may be referred to asautofluorescence or natural fluorescence. These residual fluorescentcharacteristics are generally weak signals and may have a wide spectrumof excitation and emission wavelengths. In the context of the presentdescription, these materials include certain plastics or metals presentin the system, or designed into the system. For example, MRDs of thepresent disclosure may be comprised of a polymeric material that hasresidual fluorescent characteristics. Similarly, portions of anincubator, spokes on a carousel, fiber optic materials such as plasticor glass fibers, or a component of another transport mechanism may becomprised of a material that has residual fluorescent characteristics.In certain embodiments where fluorometers are being monitored forfailure, these materials having residual fluorescence characteristicscan comprise the fluorescent reference standards of the presentdisclosure. Generally, however, the weaker residual fluorescent signalmust have enough strength to be identified by the fluorometer to beeffective as a fluorescent reference standard. Frequently this weakerresidual fluorescent signal is strong enough for detection withouthaving to adjust the strength of the excitation signal or the gain inthe system that are used to evaluate test samples. The appropriatematerials are determined for each fluorometer channel based on theexcitation and emission characteristics of the material. In theseembodiments, each fluorometer channel (e.g., FAM, ROX, HEX, etc.) mayrequire a different fluorescent reference standard, but two or morechannels may be able to share a single material if the emissioncharacteristics of the material are sufficiently distinguishable usingthe different excitation wavelengths. For example, one fluorometerchannel may use an MRD as its fluorescent reference standard, anotherfluorometer channel in the system may utilize a spoke as its fluorescentreference standard, and still another fluorometer channel may utilize ahole or indentation drilled in a spoke as its fluorescent referencestandard. In another embodiment, fiber optic materials such as fibersutilized to carry an excitation and/or emission signal (e.g., lightpipes) may be utilized as the fluorescent reference standard. Thoughplastic fiber optic materials are known to have higher levels ofautofluorescence, in general the fiber optic materials, e.g., plastic,glass, or another type of fiber material, may be chosen based on itsautofluorescence characteristics and the fluorometer requirements in thesystem. One of skill in the art can adjust the choice of fluorescentreference standards based on excitation and emission wavelengths, andsignal strengths based on the disclosure provided herein.

In one embodiment, the reference standards 250 of each referencestandard set are arranged in a linear configuration on one outer spoke264. As shown in FIG. 15, however, in the illustrated embodiment, theouter spokes 264 are not arranged in a radial orientation with respectto the lower disk 256. Each signal detector 400 is parallel to the outerspokes 264 and is oriented such that each of its five channels willsimultaneously align with one of the receptacle vessels 162 of a MRD 160positioned above the signal detector 400. Accordingly, because the MRD160 is carried within the carousel at a position that is adjacent to andparallel with the outer spoke 264, and because the outer spoke and theMRD are not carried in a radial orientation, the five referencestandards 250 of each reference set will not simultaneously align withthe five channels of a signal detector 400. Therefore, to place each ofthe reference standards 250 in optical communication with itscorresponding channel of the signal detector 400, the carousel must berotated in five increments to test all five channels of the signaldetector. In one embodiment, the carousel must be rotated 4.65 degreesfrom the position shown in FIG. 15 to place the first reference standard(i.e., the radially outermost reference standard) into opticalcommunication with the outermost channel of the signal detector. Eachsubsequent incremental rotation required to place the next fourreference standards into optical communication with its correspondingchannel are shown in the following table.

Target-spoke position Angle MRD and fluorometer aligned 0°   (as in FIG.15) Reference target aligned with 4.65° outermost fluorometer channelReference target aligned with 5.35° second fluorometer channel Referencetarget aligned with 6.25° third fluorometer channel Reference targetaligned with 7.6°  fourth fluorometer channel Reference target alignedwith fifth 9.75° (innermost) fluorometer channel

Rather than moving the receptacle carrier 242 by each of the smallincremental rotations listed above to sequentially place all thechannels of all the signal detectors 400 into self-check positions withrespect to an associated reference standard 250, in one embodiment, adifferent channel of each of the signal detectors 400 is self-testedwith each revolution of the carousel. Thus, after one revolution of thereceptacle carrier 242 during which all receptacle vessels of all theMRDs 160 are interrogated by all the signal detectors 400, the carouselis advanced until one of the channels (e.g., the first channel) of allthe signal detectors 400 is aligned with an associated referencestandard 250 for a self-check of all the first channels. After a nextrevolution of the receptacle carrier 242, the carousel is advanced untilthe next channel (e.g., the second) of all the signal detectors 400 isaligned with an associated reference standard 250 for a self-check ofall the second channels. This process is repeated for each subsequentrevolution of the receptacle carrier 242 to self-check the third,fourth, and fifth channels of the signal detectors 400. Thus, accordingto this embodiment, each channel of each signal detector is self-checkedonce in every five revolutions of the carousel.

In one embodiment, the signal detectors 400 are configured so that areceptacle vessel 162 carried on the receptacle carrier 242 will be atan optical focal point of the signal detector 400 when the receptaclevessel 162 is operatively positioned above the signal detector 400. Inone embodiment, however, the bottom of each receptacle vessel 162 ispositioned above the lower disk 256 on which the reference standards 250are mounted. Thus, in this embodiment the reference standards 250 arenot at the optical focal point of the signal detectors 400. This isillustrated in FIGS. 16 and 17. As shown in FIG. 16, when the receptaclevessel 162 is positioned above the signal detector 400 within theincubator 200, a portion of the receptacle vessel 162 (e.g.,approximately within the contents of the receptacle vessel 162) is at afocal point f, or detection zone, at a height h₂ with respect to thecorresponding channel of the signal detector 400. The reference standard250, on the other hand, mounted to the outer spoke 264 of the lower disk256 is at a height h₁ that is less than h₂ above the signal detector400. In one embodiment, h₁ is 1.0 mm and h₂ is 8.5 mm. In anotherembodiment, h₁ is 2.0 mm and h₂ is 10 mm. In other embodiments, h₁ is 1%to 99% smaller than h₂, in other embodiments, h₁ is 20% to 80% smallerthan h₂, and in still other embodiments, h₁ is 60% to 90% smaller thanh₂. Thus, as shown in FIG. 17, after the receptacle carrier 242 rotatesin the direction of the lateral arrow to move the receptacle vessel 162out of the focal point (or detection zone) and place the referencestandard 250 into optical communication with respect to the signaldetector 400, the reference standard 250 is not at the focal point(i.e., is not in the detection zone) of the corresponding channel of thesignal detector 400. Nevertheless, even with the fluorescent referencestandards 250 out of focus with respect to the signal detector 400, theinventors have discovered that if the optical emission from thereference standard is suitably robust—meaning that the emissionintensity of the reference standard is more intense than the emissionintensity of the sample contained within the receptacle vessel 162,which is in focus with respect to the fluorometer—changes in theemission signal from the reference standard due to failure orperformance deterioration of the fluorometer can still be adequatelydetected.

In other embodiments, h₂ is less than h₁. That is, the support structureon which the reference standards are mounted may be above (or otherwisefurther from) the focal point f at h₂. In such embodiments, h₂ may be 1%to 99% smaller than h₁, 20% to 80% smaller than h₁, or 60% to 90%smaller than h₁.

With the reference standard out of focus with respect to thefluorometer, calibrating the fluorometer with the reference standard maybe complicated, because it is difficult to obtain precise fluorescentemissions signals from an out of focus reference standard. Thus, themeasured fluorescent emission signal from an out of focus referencestandard will not frequently be compared to an expected emission signalfor the purpose of calibrating the fluorometer. There is likely to betoo much variability in measured emission intensity from one measurementto the next. On the other hand, as explained above, even with thereference standard out of focus with respect to the fluorometer, if thereference standard has sufficient emission intensity, it is stillpossible to confirm proper functioning of the fluorometer by detectingan emission signal of at least a specified intensity level.

In other embodiments, reference standards can be mounted in positionsthat are in focus with respect to the fluorometer (or the fluorometercan be configured so that the focal point is adjustable) so that precisereference standard emission signals can be obtained and compared toexpected signals so that the fluorometer can be calibrated based on themeasured reference standard emission signal.

As represented schematically in FIG. 17, the focal cone which terminatesat the apex f actually overfills the reference standard 250 at heighth₁. Thus, reference readings are sensitive to the horizontal positioningof the reference standard 250 because lateral, horizontal movement ofthe reference standard 250 can cause all or a portion of the referencestandard 250 to be positioned outside the focal cone. Thus, incircumstances, such as shown in FIG. 17, in which the focal coneoverfills the reference standard 250, positional accuracy of the supporton which the reference standard 250 is mounted (e.g., the receptaclecarrier 242) is important.

An automated, self-check procedure for a signal detector 400 isrepresented by flow chart 350 shown in FIG. 18. The procedure isperformed with the signal detector 400 and the receptacle carrier 242,which are controlled by a computer controller (microprocessor) executingsoftware that includes an algorithm embodying procedure 350 encoded orstored on a computer-readable medium.

At Step 352, an initial, or baseline, reference reading is establishedfor each channel (j) for each signal detector, or fluorometer, (k) bymoving the reference standard into optical communication with thechannel and measuring an initial optical emission intensity from anassociated optical reference standard. Using the example of fluorometersand fluorescent reference standards, a quantifying unit of fluorescentemission intensity may be referred to as a “Relative Fluorescent Unit”(“RFU”), and the initial reference intensity of the j^(th) channel forthe k^(th) fluorometer is RFU_(Ijk). The initial, or baseline, referenceintensity RFU_(Ijk) can be measured before the signal detector 400 isinstalled in the incubator 200 or after the fluorometer is installed inthe incubator.

At Step 354, the initial reference reading for each channel for eachfluorometer (RFU_(Ijk)) taken at Step 352 is stored in suitable memorythat is accessible by a microprocessor controller.

At Step 356, after an interval of usage of the fluorometer, a testreference reading is taken for each channel (j) for each fluorometer (k)by moving the associated reference standard into optical communicationwith the channel and measuring an optical emission intensity from theassociated optical reference standard. The test reference reading ispreferably taken from the same optical reference standard from which theinitial reading was taken, since the fluorescent emissions ofconsumer-grade fluorescent plastics can vary significantly from batch tobatch, even for the same nominal color. The test reference intensity ofthe j^(th) channel for the k^(th) fluorometer is RFU_(Tjk). In thisregard, if a reference standard is replaced after a fluorometer has beenplaced in service; it is preferable to obtain a new initial referencereading for the replaced reference standard. Obtaining a new initialreference reading is important due to the operational lifespan of afluorometer, which may deteriorate gradually over time in its excitationsignal intensity. As such, the new initial reference reading willaccount for the excitation signal deterioration, yet provide a usefulmeasure of the operational capacity of the fluorometer (i.e., determineif a failure has occurred).

The typical lifespan of fluorometers that are currently available on themarket is, depending on usage frequency and duration, about 5 to 7years, or about 50,000 hours of operation. Of course, one of skill inthe art would appreciate that fluorometer lifespan will vary dependingon model, manufacturer, and usage conditions. Over this time LEDintensity can, and often does, decrease. The present methods areeffective in monitoring the performance of the fluorometer during thisgradual decrease in signal intensity witnessed over extended periods oftime. For example, Tables 2 and 3 below provide RFU readings of AcrycastCell Cast Acrylic sheet part No. 2422 color Amber plastic on FAM, HEXand ROX fluorometers (Table 2) and RFU readings of McMaster amberplastic 3.5 mm disks on ROX fluorometers (Table 3).

TABLE 2 Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 No. 5 FAM RTF@20 mA 7700 6900 8200 8400 8700 No. 5 FAM RTF @40 mA 13400 12200 1450014800 15000 Decrease from 40 to 20 MA 42.54% 43.44% 43.45% 43.24% 42.00%No. 10 FAM RTF @20 mA 8700 10300 9400 10600 9700 No. 10 FAM RTF @40 mA15200 18400 16800 19100 17100 Decrease from 40 to 20 MA 42.76% 44.02%44.05% 44.50% 43.27% No. 5 HEX RTF @20 mA 4500 6500 6100 7900 6900 No. 5HEX RTF @40 mA 7700 10700 10300 13000 10700 Decrease from 40 to 20 MA41.56% 39.25% 40.78% 39.23% 35.51% No. 10 HEX HEX @20 mA 7000 6900 77008300 8100 No. 10 HEX HEX @40 mA 9950 9850 11300 12200 12100 Decreasefrom 40 to 20 MA 29.65% 29.95% 31.86% 31.97% 33.06%

TABLE 3 Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 No. 5 ROX RTF@20 mA 8200 7600 6600 10100 7600 No. 5 ROX RTF @40 mA 12900 12000 1030015900 12100 Decrease from 40 to 20 MA 36.43% 36.67% 35.92% 36.48% 37.19%No. 10 ROX RTF @20 mA 9600 10800 7700 8300 8200 No. 10 ROX RTF @40 mA17000 19000 13700 14700 14600 Decrease from 40 to 20 MA 43.53% 43.16%43.80% 43.54% 43.84%

Each channel was tested at two different currents (i.e., 20 mA and 40mA) to approximate the downward shift in signal intensity that iswitnessed over time with a fluorometer. Though a measureable decrease inemission signal intensity with decrease in excitation intensity wasobserved across all channels, significant levels of fluorescenceemissions were detected at the lower current level across all testedfluorophores and fluorometers.

At Step 358, the initial reference reading for the j^(th) channel andthe k^(th) fluorometer (RFU_(Ijk)) is retrieved from storage, and, atStep 360, the test reference reading is compared to the initialreference reading. In one embodiment, Step 360 is performed by analgorithm that computes the difference between the test referencereading and the initial reference reading for the channel, and in Step362 the absolute value of that difference is compared to a predeterminedthreshold value. In one embodiment, a suitable threshold is within a 30%deviation from the initial reference reading. In another embodiment, asuitable threshold is within a 40%-99% deviation from the initialreference reading.

If the absolute value of the difference between the test and initialreference readings is at the threshold or higher, a possible malfunctionof the fluorometer is indicated, and, per Step 364 an error warning orother indication of the possible malfunction is provided, and operationof the fluorometer may be interrupted or terminated.

If the absolute value of the difference between the test and initialreference readings is below the threshold, the fluorometer is deemed tobe functioning properly, and, per Step 366, operation continues and, aslong as the fluorometer continues operation (until a stop condition isreached), periodic self-checks are performed by repeating Steps 356,358, 360, and 362.

In one embodiment, it is preferred that periodic test reference readingsbe taken and compared to the initial reference readings at least onceevery 50 minutes, though the interval can vary significantly inaccordance with the user preferences and type of assay being performed.A stop condition may be indicated by completion of the test or assay, aneed to stop operation of the instrument to replenish reagents, MRDs, orother disposables, or if, during a fluorometer self-check, the deviationbetween the test and initial, or baseline, reference readings exceedsthe threshold.

Steps 358, 360, 362, 364, and 366 are performed by the computercontroller (microprocessor) executing software that includes analgorithm embodying Steps 358, 360, 362, 364, and 366 encoded or storedon a computer-readable medium.

If reference readings are taken every 50 minutes, as mentioned above,and the instrument is operated 12 hours a day for 300 days per year,25,920 reference readings will be taken after six years of operation.This is equivalent to 14.4 hours of continuous testing at a 2 secondsampling rate. Thus, it is important that a reference standard materialbe selected that is not vulnerable to photo bleaching.

The process steps of an exemplary real-time amplification assayprocedure 1900 are illustrated in the flow chart shown in FIG. 27. Theprocedure 1900 is performed by a diagnostic analyzer of which one ormore incubators, such as incubator 200, is a component and which iscontrolled by a computer (microprocessor) executing software thatincludes an algorithm embodying procedure 1900 encoded or stored on acomputer-readable medium. The process shown in FIG. 27 is similar to ananalogous process described in detail in Macioszek et al., “Methods forPerforming Multi-Formatted assays,” U.S. Pat. No. 7,897,337. The stepsdescribed represent exemplary TAA procedures only. Persons of ordinaryskill will recognize that the steps described below may be varied oromitted or that other steps may be added or substituted in accordancewith other real-time amplification assay procedures now known or yet tobe developed. Reagent formulations for performing a host ofamplification procedures are well known in the art and could be used inor readily adapted for use in the present invention. See, e.g., Kacianet al., U.S. Pat. No. 5,399,491; Becker et al., U.S. Pat. No. 7,374,885;Linnen et al., Compositions and Methods for Detecting West Nile Virus,U.S. Pat. No. 7,115,374; Weisburg et al., “Compositions, Methods andKits for Determining the Presence of Trichomonas Vaginalis in a TestSample,” U.S. Pat. No. 7,381,811; and Kacian, “Methods for Determiningthe Presence of SARS Coronavirus in a Sample,” U.S. Patent ApplicationPublication No. 2010-0279276 A1.

The process steps of the exemplary real-time TAA amplification assaybegin with step 1902, in which a receptacle, such as an MRD 160, ismoved to a pipetting position in a sample transfer station (not shown).In step 1904, a sample pipette assembly (not shown) dispenses 100 μL ofa target capture reagent (“TCR”) including magnetically-responsiveparticles into the receptacle, e.g., into each receptacle vessel 162 ofthe MRD 160. The target capture reagent includes a capture probe, adetergent-containing lytic agent, such as lithium lauryl sulfate, forlysing cells and inhibiting the activity of RNAses present in the samplematerial, and about 40 μg Sera-Mag™ MG-CM Carboxylate Modified (Seradyn,Inc., Indianapolis, Ind.; Cat. No. 24152105-050250), 1 micron,super-paramagnetic particles having a covalently bound poly(dT)14. Thecapture probe includes a 5′ target binding region and a 3′ region havinga poly(dA)30 tail for binding to the poly(dT)14 bound to the magneticparticle. The target binding region of the capture probe is designed tobind to a region of the target nucleic acid distinct from the regionstargeted by the primers and the detection probe.

In step 1906, 400 μL of sample is dispensed into the receptacle. In step1908, the receptacle, e.g., MRD 160, is moved to a mixer (not shown),and in step 1909, the sample and TCR are mixed, preferably at 16 Hz for60 seconds. Note that the times given in FIG. 27 and the descriptionthereof are desired times, and the actual times may, in practice, varyfrom the given desired times.

In one embodiment, the diagnostic analyzer includes three incubatorsmaintained at three different temperatures: a first incubator maintainedat 64° C. for target capture and primer annealing, a second incubatormaintained at 43.7° C. for pre-heating receptacles, AT binding, andprimer binding, and a third incubator maintained at 42.7° C. foramplification. The first, second, and third incubators may be configuredthe same as incubator 200 described above, although the first and secondincubators may omit the signal detectors 400.

In step 1910, the receptacle is moved to the second incubator topre-heat the receptacle and its contents at a temperature of 43.7° C.for 276 seconds. In other embodiments, the receptacle may be placed in atemperature ramping station (i.e., a temperature-controlled enclosure(not shown) configured to receive and hold one or more receptacles) forthe pre-heating step. In step 1912, the receptacle is moved to the firstincubator (i.e., target capture (“TC”) incubator) where it resides at64° C. for 1701 seconds for hybridization of the capture probe to targetnucleic acids extracted from the sample. (At this temperature, therewill be no appreciable hybridization of the capture probe to theimmobilized poly(dT)14 oligonucleotide.) In step 1914, the receptacle ismoved from the TC incubator to the second incubator for AT binding whereit is held for 313 seconds at 43.7° C. to allow for immobilizedoligonucleotides associated with the magnetic particles to bind to thecapture probes. In step 1916, the receptacle is moved to a coolingchiller (i.e., a temperature-controlled enclosure configured to receiveand hold one or more receptacles (not shown)) where the receptacle isheld at 18° C. for 481 seconds.

In step 1918, the receptacle is moved to a magnetic parking station (notshown), which is a structure configured to hold one or more receptaclesin proximity to one or more magnets so that the contents of eachreceptacle vessel 162 are exposed to a magnetic field to draw themagnetically-responsive particles of the target capture reagent to aportion of the receptacle adjacent to the magnet and out of suspension.A suitable magnetic parking station is described in Davis, et al., U.S.Patent Application Publication No. 2010/0294047, “Method and System forPerforming a Magnetic Separation Procedure.”

In step 1920, the receptacle is moved to a magnetic separation station(not shown) for the magnetic separation wash procedure, such as isdescribed in Lair et al., U.S. Patent Application Publication No.2007-0243600 A1. Within the magnetic separation station, magnets, whichare selectively placed in close proximity to the reaction vessel, areused to draw and hold the magnetically-responsive particles to a portionof the vessel. Once the magnetically-responsive particles, and anytarget nucleic acid bound thereto, are thus immobilized, the hybridizednucleic acid can be separated from non-hybridized nucleic acid byaspirating fluid from the reaction vessel. After the initial aspirationof the fluid contents from the vessel, 1 mL of wash solution is added tothe receptacle in step 1922. Step 1924 comprises a second magnetic wash,which includes, after the fluid contents of the receptacle areaspirated, adding 1 mL wash solution to the receptacle in step 1926 andadding 100 μL oil (e.g., silicone oil), or other surface treating agent,to the receptacle in step 1928. In step 1930, a final magnetic washprocedure is performed (in other embodiments, more or fewer magneticwash procedures can be performed) followed by a final dispense of 100 μLoil (e.g., silicone oil), or other surface treatment agent, in step1932.

An advantage of adding a surface treating agent, such as silicone oil,to the sample solution in steps 1928 is that it reduces the amount ofmaterial that adheres to the inner surfaces of the reaction vessels 162during the rinsing and aspiration steps of a magnetic separation washprocedure, thereby facilitating a more effective magnetic separationwash procedure. Although the MRDs 160 are preferably made of ahydrophobic material, such as polypropylene, small droplets of material,such as wash solution, may still form on the inner surfaces of the MRDreceptacle vessels 162 during the aspiration steps of a magneticseparation wash procedure. If not adequately removed from the receptaclevessels 162 during the magnetic separation wash procedure, this residualmaterial, which may contain nucleic acid amplification inhibitors, couldaffect assay results. In alternative approaches, the surface treatingreagent could be added to the receptacle vessels 162 and removed priorto adding TCR and sample or the surface treating agent could be added tothe reaction tubes after TCR and sample have been aspirated from thereaction tubes, possibly with the wash solution, and then removed priorto adding amplification and enzyme reagents to the reaction tubes. Theobjective is to provide inner surfaces of the receptacle vessels 162with a coating of the surface treating agent. Inhibitors ofamplification reactions are known in the art and depend on the samplesource and amplification procedure to being used. Possible amplificationinhibitors include the following: hemoglobin from blood samples;hemoglobin, nitrates, crystals and/or beta-human chorionic gonadotropinfrom urine samples; nucleases; proteases; anionic detergents such assodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS); and EDTA,which is an anticoagulant and fixative of some specimens that bindsdivalent cations like magnesium, which, as noted above, is a cofactorused in nucleic acid-based amplification reactions. See, e.g., Mahony etal., J. Clin. Microbiol., 36(11):3122-2126 (1998); Al-Soud, J. Clin.Microbiol., 39(2):485-493 (2001); and Kacian et al., “Method forSuppressing Inhibition of Enzyme-Mediated Reactions By Ionic DetergentsUsing High Concentration of Non-Ionic Detergent,” U.S. Pat. No.5,846,701. Silicone oil is added to each reaction vessel 162 of the MRD160 in step 1932 to prevent evaporation and splashing of the fluidcontents during subsequent manipulations.

In step 1934, amplification reagent, which is stored in a chilledenvironment, is added to each receptacle while the receptacle is held at45° C. at an amplification load station (not shown). In step 1936, 75 μLof an amplification reagent are dispensed into the receptacle disposedwithin the load station, and the receptacle is then mixed for 25 secondsat 16 Hz by a mixer incorporated into the load station. For theexemplary TAA reactions, the amplification reagents contain an antisensepromoter-primer having a 3′ target binding region and a 5′ promotersequence recognized by an RNA polymerase, a sense primer that binds toan extension product formed with the promoter-primer, nucleosidetriphosphates (i.e., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), andcofactors sufficient to perform a TAA reaction. For the real-time TAAamplification assay, the amplification reagent also contains stranddisplacement, molecular torch probes having interacting label pairs(e.g., interacting fluorescent and quencher moieties joined to the 5′and 3′ ends thereof by conventional means) and a target specific regioncapable of detectably hybridizing to amplification products as theamplification is occurring and, preferably, not to any non-targetnucleic acids which may be present in the receptacles. See Kacian etal., U.S. Pat. No. 5,399,491; Becker et al., “Single-Primer Nucleic AcidAmplification,” U.S. Pat. No. 7,374,885 (disclosing an alternativeTAA-based amplification assay in which an antisense primer and a sensepromoter oligonucleotide blocked at its 3′ end are employed to minimizeside-product formation); and Becker et al., U.S. Pat. No. 6,361,945.

In step 1938, the receptacle is moved to the second incubator andpreheated at 43.7° C. for 286 sec. In step 1940, the receptacle is movedto the first incubator and incubated at 64° C. for 636 seconds forprimer annealing. In step 1942, the receptacle is moved to the secondincubator and incubated for 405 seconds at 43.7° C. for binding of thepromoter-primer to a target nucleic acid. The preferred promoter-primerin this particular TAA example has a promoter sequence recognized by aT7 RNA polymerase.

In step 1944, the receptacle is moved to the load station for enzymereagent addition at 45° C. In step 1946, 25 μL of enzyme are added andthe MRD is mixed at 10 Hz for 15 seconds. In step 1948, the receptacleis moved to the third incubator (amplification incubator), where thereceptacle contents are incubated at 42.7° C. for 3569 seconds foramplification. During amplification, real-time fluorescence measurementsare taken in step 1950. In one embodiment, step 1950 comprises takingmultiple, real-time fluorescence measurements during rotation of thecarousel 242 whereby each receptacle vessel 162 of each MRD 160 isinterrogated by each signal detector 400 once per revolution of thecarousel 242. During step 1950, each channel of each signal detector 400is periodically self-checked, e.g., once every five revolutions of thecarousel 242 as described above, using steps 356 to 366 of the automatedself-check procedure 350 shown in FIG. 18. The enzyme reagent of thisexample contains a reverse transcriptase and a T7 RNA polymerase forperforming TAA.

After the nucleic acid-based assay is complete, and to avoid possiblecontamination of subsequent amplification reactions, the reactionmixture can be treated with a deactivating reagent which destroysnucleic acids and related amplification products in the reaction vessel.In such an example, following amplification and real-time measurements,in step 1952, the receptacle is moved to a deactivation queue, or module(not shown), and, in step 1954, 2 mL of a bleach-based agent areprovided to each receptacle to deactivate nucleic acid (i.e., alter thenucleic acid such that it is non-amplifiable) present in the receptacle.Such deactivating agents can include oxidants, reductants and reactivechemicals, among others, which modify the primary chemical structure ofa nucleic acid. These reagents operate by rendering nucleic acids inerttowards an amplification reaction, whether the nucleic acid is RNA orDNA. Examples of such chemical agents include solutions of sodiumhypochlorite (bleach), solutions of potassium permanganate, formic acid,hydrazine, dimethyl sulfate and similar compounds. More details of adeactivation protocol can be found in, e.g., Dattagupta et al., U.S.Pat. No. 5,612,200, and Nelson et al., U.S. Patent ApplicationPublication No. US 2005-0202491 A1.

As noted above, the incubator 200 includes a number of signal detectors400 configured to measure in real time the concentration of unquenchedfluorescent dye molecules located in the MRD 160. As discussed above,the assay is designed such that the fluorescent signal increases as theconcentration of the target is increased by amplification. The detectors400, therefore, may be used to monitor the amplification process bymonitoring the emergence of the fluorescent signal.

Once the data has been collected by measuring fluorometric emissionsfrom each receptacle at prescribed intervals for a prescribed period oftime, and while periodically self-checking the fluorometer as describedabove to confirm that the fluorometer is functioning properly, the datais processed to determine the concentration of a particular analyte(e.g., target nucleic acid) in the sample. The measured data, that is,the measured signal, will be referred to in terms of a RelativeFluorescent Unit (“RFU”), which is the signal generated by the detectionPCB 422 of the signal detector 400 based on the amount of emissionfluorescence focused onto the detection element 423. Each data point,measured at a given time interval, is RFU(t). Plots of RFU(t) for avariety of data sets, known as “growth curves” are shown in FIG. 29. Ingeneral, each RFU(t) plot is generally sigmoidal in shape, characterizedby an initial, flat portion (known as the “static level” or “baselinephase”) at or near a minimum level, followed by an abrupt and relativelysteeply sloped portion (known as the “growth phase”), and ending with agenerally flat portion at or near a maximum level (known as the “plateauphase”).

As used herein, a “growth curve” refers to the characteristic pattern ofappearance of a synthetic product, such as an amplicon, in a reaction asa function of time or cycle number. A growth curve is convenientlyrepresented as a two-dimensional plot of time (x-axis) against someindicator of product amount, such as a fluorescence measurement—RFU(y-axis). Some, but not all, growth curves have a sigmoid-shape. The“baseline phase” of a growth curve refers to the initial phase of thecurve wherein the amount of product (such as an amplicon) increases at asubstantially constant rate, this rate being less than the rate ofincrease characteristic of the growth phase (which may have a log-linearprofile) of the growth curve. The baseline phase of a growth curvetypically has a very shallow slope, frequently approximating zero. The“growth phase” of a growth curve refers to the portion of the curvewherein the measurable product substantially increases with time.Transition from the baseline phase into the growth phase in a typicalnucleic acid amplification reaction is characterized by the appearanceof amplicon at a rate that increases with time. Transition from thegrowth phase to the plateau phase of the growth curve begins at aninflection point where the rate of amplicon appearance begins todecrease. The “plateau phase” refers to the final phase of the curve. Inthe plateau phase, the rate of measurable product formation issubstantially lower than the rate of amplicon production in thelog-linear growth phase, and may even approach zero.

A process for calculating an analyte concentration is shown by means ofa flow chart in FIG. 28. The RFU(t) data from the signal detector 400 isinput as represented at box 2100. The RFU(t) data goes to threshold timedetermination, which begins at 2104. Threshold time, or T-time, (alsoknown as time of emergence) refers to the time at which the data RFU(t),normalized as discussed below, reaches a predefined threshold value.Using calibration curves, as will be described in more detail below, theT-time determined for a particular sample can be correlated with ananalyte concentration, thereby indicating the analyte concentration forthe sample. In general, the higher the concentration of the analyte ofinterest, the sooner the T-time is reached.

The first step of the T-time determination procedure is backgroundadjustment and normalization of the data, as represented at box 2106.Background adjustment is performed to subtract that portion of thesignal data RFU(t) that is due to background “noise” from, for example,stray electromagnetic signals. That is, the background noise includesthat part of the RFU(t) signal due to sources other than the analyte ofinterest. Background adjustment is performed by subtracting a backgroundvalue “BG” from the data RFU(t) to obtain adjusted data RFU*(t). Thatis, RFU*(t)=RFU(t)−BG.

The background BG can be determined in a number of ways.

In accordance with one method for determining the background noise, thefirst step is to determine the time intervals between data points. Thetime interval is determined by multiplying cycle time (i.e., the timebetween consecutive data measurements) by the data point (i.e., 0^(th)data point, 1^(st) data point, 2^(nd) data point, . . . , n^(th) datapoint) and divide by 60 seconds. For example, assuming a cycle time of30 seconds, the time interval for the 15^(th) data point is (15×30sec.)/60 sec.=7.5.

The next step is to find the midpoint of the signal data by adding theminimum signal data point and the maximum signal data point and dividingby two. That is:(RFU_(max)+RFU_(min))/2.

Starting at the time corresponding to the midpoint value and workingbackwards, calculate the slope for each pair of data points:(RFU(t)−RFU(t−1))/Δt(t→t−1).

Next, determine where the slope of RFU(t) flattens out by finding thefirst slope value that is less than the static slope value (i.e., thevalue before the RFU(t) curve begins its upward slope). A representativestatic slope value, also known as the “delta value,” includes 0.0001.Once this slope is found, find the next cycle in which the slope that isnot negative or is, for example, above the negative delta value (i.e.,−0.0001); this value is H_(index). Next, take the mean of the entirerange of RFU(t) values starting at the first data point and go to theRFU value that corresponds to the H_(index) value. The mean of this datamay be computed using the Excel TRIMMEAN function on this range of datausing a static back trim value of 0.15 (that is, the lowest 7.5% of RFUvalues in the specified range and the highest 7.5% RFU values in thespecified range are excluded). This mean value is the background, BG.Alternatively, the background can be determined in accordance with theprocedure described above using a delta value other than 0.0001.

A further alternative method for determining the background eliminatesthe delta value criterion and instead take a TRIMMEAN mean of the RFUdata from cycle 1 to a prescribed end point, such as the first cyclebefore 5.5 minutes. For this alternative, the static back trim value maybe adjusted to, for example, 0.40 (that is, the lowest 20% of RFU valuesin the specified range and the highest 20% RFU values in the specifiedrange are excluded from the background calculation).

A further alternative method for determining the background is toperform a curve fit on all or a portion of the RFU data to derive anestimate of the baseline value, which is the background to besubtracted. Any curve fit technique suitable for fitting a curve to theRFU data can be used.

An exemplary curve fit technique is to use a portion of the equationderived by Weusten et al. for curve fit of the typically sigmoidalcurves associated with nucleic acid amplification. See Weusten et al.,Nucleic Acids Research, 30(6e26):1-7 (2002). For background subtraction,it is only necessary to ascertain the baseline level. Thus, it is alsoonly necessary to fit a curve to the first portion of the RFU dataencompassing the baseline, usually toward the beginning of the curve.

The curve fit may be performed on the RFU(t) data from cycle 1 to thecycle just before 75% of the maximum RFU. The following polynomialequation (3), which, as mentioned above, is a portion of the equationderived by Weusten et al, is used to generate a best fit model of theRFU data:RFU(t)=Y0+a1a2[e ^(a2(t-a3))/(1+e ^(a2(t-a3)))] ln(1+e ^(a2(t-a3)))  (3)

Initial estimates for the variables Y0, a1, a2, and a3, as discussedbelow, are input to the curve-fit equation and an iterative solutionfitting the equation to the RFU data is performed, for example, usingthe SOLVER function of Microsoft EXCEL, to yield the final equation andthe final values for Y0, a1, a2, and a3.

Y0=is the baseline; an initial value can be RFU(1).

a1=relates to the steep portion (growth phase) of the RFU(t) data; 0.05can be a suitable initial estimate for a1.

a2=relates to the steep portion (growth phase) of the RFU(t) data; 1.0can be a suitable initial estimate for a2.

a3=relates to the transition between the baseline and the slope feature;the time, or cycle, at which RFU(t) reaches a value just before 25% ofRFU_(max) is a suitable initial estimate for a3.

When the final values of Y0, a1, a2, and a3 have been derived, Y0 istreated as the back ground, and is subtracted from the RFU(t) data forwhich the curve fit was performed.

Curve fit equations other than that described above can be used. Forexample, the commercially available TABLECURVE software package (SYSTATSoftware Inc.; Richmond, Calif.) can be used to identify and selectequations that describe exemplary real-time nucleic acid amplificationcurves. One such exemplary resulting equation, used for mathematicalmodeling, is given by equation (4):RFU(t)=Y0+b(1−exp(−(t−d*n(1−2{circumflex over( )}^((-1/e)))−c)/d)){circumflex over ( )}^(e)  (4)Still another exemplary resulting equation is given by equation (5):RFU(t)=Y0+b/(1+exp(−(t−d*ln(2{circumflex over( )}^((1/e))−1)−c)/d)){circumflex over ( )}^(e)  (5)

In each case, as described above, the equation can be solved, forexample, using the SOLVER function of Microsoft EXCEL, to yield thefinal equation and the final values for Y0 and the other parameters, andthe solutions yields a Y0 that is the background to be subtracted fromthe RFU(t) data.

To normalize the data, each data point, adjusted for the background, isdivided by the maximum data point, also adjusted for the background.That is:

${{Normalized}\mspace{14mu}{RFU}} = {{{RFU}_{n}(t)} = {\frac{{RFU}^{*}(t)}{{RFU}_{\max}^{*}} = \frac{\left( {{{RFU}(t)} - {BG}} \right)}{\left( {{RFU}_{\max} - {BG}} \right)}}}$Thus, the RFU_(n)(t) will be from −1 to 1.

In step 2108, the range of data is calculated by subtractingRFU_(n(min)) from RFU_(n(max)). If the calculated range does not meet orexceed a specified, minimum range (e.g., 0.05), the data is consideredsuspect and of questionable reliability, and, thus, the T-time will notbe calculated. The minimum range is determined empirically and may varyfrom one fluorescence measuring instrument to the next. Ideally, thespecified minimum range is selected to ensure that the variation of datavalues from minimum to maximum exceeds the noise of the system.

In step 2110, a curve fit procedure is applied to the normalized,background-adjusted data. Although any of the well-known curve fitmethodologies may be employed, in a preferred embodiment, a linear leastsquares (“LLS”) curve fit is employed. The curve fit is performed foronly a portion of the data between a predetermined low bound and highbound. The ultimate goal, after finding the curve that fits the data, isto find the time corresponding to the point at which the curveintersects a predefined threshold value. In the preferred embodiment,the threshold for normalized data is 0.11. The high and low bounds aredetermined empirically by fitting curves to a variety of control datasets and observing the time at which the various curves cross the chosenthreshold. The high and low bounds define the upper and lower ends,respectively, of the range of data over which the curves exhibit theleast variability in the times at which the curves cross the giventhreshold value. In the preferred embodiment, the low bound is 0.04 andthe high bound is 0.36—See FIG. 29. The curve is fit for data extendingfrom the first data point below the low bound through the first datapoint past the high bound.

At step 2112, determine whether the slope of the fit is statisticallysignificant. For example, if the p value of the first order coefficientis less than 0.05, the fit is considered significant, and processingcontinues. If not, processing stops. Alternatively, the validity of thedata can be determined by the R2 value.

The slope m and intercept b of the linear curve y=mx+b are determinedfor the fitted curve. With that information, T-time can be determined atstep 2114 as follows:

${T\text{-}{time}} = \frac{{Threshold} - b}{m}$The technique of using the fitted curve to determine T-times isillustrated graphically in FIG. 30.

Returning to FIG. 28, at step 2116, it is determined whether or notinternal control/calibrator adjustments are desired. Typically, a testprocedure would include at least one reaction vessel with a knownconcentration of a nucleic acid (other than a nucleic acid of interest)as a control, or, alternatively, a control nucleic acid sequence can beadded to each sample. The known concentration can be simply used ascontrol to confirm that a reaction did take place in the reactionvessel. That is, if the known concentration is amplified as expected,successful reaction is confirmed and a negative result with respect tothe target analyte is concluded to be due to absence of target in thesample. On the other hand, failure to amplify the known concentration asexpected indicates a failure of the reaction and any result with respectto the target is ignored.

The known concentration can be used to calibrate the concentration ofthe target at step 2118. The T-times corresponding to a series ofstandards containing internal control and target sequences aredetermined for a statistically valid number of data sets. Using thisdata, a calibration plot is constructed from which the test sample'sconcentration is interpolated as described below.

One method of constructing the calibration plot places the knownconcentrations of target analyte on the x-axis versus the differencebetween target and control T-times on the y-axis. Subsequently, the testsample's concentration is interpolated from the calibration curve fit.Another method of constructing the calibration plot places the knownconcentration of target analyte on the x-axis versus the fraction[target T-time/internal control T-time] on the y-axis. Subsequently, thetest sample's concentration is interpolated from the calibration curvefit. An example of this is disclosed in Haaland, et al., “Methods,Apparatus and Computer Program Products for Determining Quantities ofNucleic Acid Sequences in Samples Using Standard Curves andAmplification Ratio Estimates,” U.S. Pat. No. 6,066,458. A furtheralternative method of constructing the calibration plot utilizes aparametric calibration method, such as the method described in Carricket al., “Parametric Calibration Method,” U.S. Pat. No. 7,831,417.

Occasionally, data sets exhibit a dip just after the initial staticbaseline (i.e., the initial, flat part of the RFU(t) curve, see FIG. 29)and just before the data begins its upward slope. To identify andcorrect such data, and prior to determining the T-time for that data,the following algorithm is employed. Starting at H_(index), check eachRFU(t) value to determine if it is less than the background value, BG.If yes, subtract RFU(t) from BG (the result should be a positivenumber). This will be the CorValue. Add the CorValue to the backgroundsubtracted value, this in turn will bring RFU(t) up to the baseline.Perform this analysis working forward on each RFU(t) value until thelatest CorValue is less than the preceding CorValue. Add the greatestCorValue to each of the remaining background subtracted RFU(t) values.Now, the corrected data set can be normalized and the T-time determinedas described above.

If a curve fit method is used to derive the background level, it may notbe necessary to perform the dip correction described above. It may alsobe desirable to perform outlier detection on the data set to identifyand, if necessary, discard data points that exhibit abnormal values ascompared to the remaining data points. Any of the well-known outlierdetection methodologies can be used.

The quantitation procedure 2120 is the second part of the analyteconcentration determination. T-times are determined for knownconcentrations of analytes for known conditions. Using this data,relationships between analyte concentrations (typically expressed as logcopy) and T-times can be derived. After a T-time is determined for aparticular sample, the derived relationship (Log copy=f (T−time)) can beused to determine the analyte concentration for the sample.

More specifically, at steps 2122 and 2124, calibration/control data setsfor a control analyte of known concentrations are validated by, forexample, outlier analysis and/or any other known data validationmethodologies. If the data is found to be valid, calibration continues,otherwise, calibration stops.

T-times for the control data sets are determined, and T-time vs. Logcopy is plotted for all samples of a particular condition (e.g., samplesprocessed with reagents from a particular batch lot). In step 2126, acurve fit, such as a linear least squares fit, is performed on a portionof the T-time vs. Log copy plot to find the slope m and intercept b ofthe line that best fits the data. If the number of available T-time vs.Log copy data points (known as “calibrators”) is not less than apredefined minimum number of calibrators (as determined at step 2128),lowest calibrators, if any, are removed at step 2130, as follows:

After finding the best fit line for the calibrator data points, 2^(nd)and 3^(rd) order curve fits are tested as well. If these fits aresignificantly better than the 1st order, linear fit, the calibrator datapoint that is furthest from the linear curve fit is discarded, and1^(st), 2^(nd) and 3^(rd) fits are found and compared again with theremaining calibrators. This process is repeated—assuming that the numberof calibrators is not less than the minimum acceptable number ofcalibrators—until the 2nd and 3rd order fits are not significantlybetter than the 1^(st) order, linear fit.

When the linear T-time vs. Log copy equation has been derived, theconcentration (as Log copy) of the analyte of interest for a sample isdetermined, at step 2132, by plugging the T-time for that sample intothe equation. Thus, the assay results are obtained 2134.

All documents referred to herein are hereby incorporated by referenceherein. No document, however, is admitted to be prior art to the claimedsubject matter.

While the invention has been described in connection with what arepresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not to be limited to thedisclosed embodiments, but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

Furthermore, those of the appended claims which do not include languagein the “means for performing a specified function” format permittedunder 35 U.S.C. § 112(16), are not intended to be interpreted under 35U.S.C. § 112(16) as being limited to the structure, material, or actsdescribed in the present specification and their equivalents.

The invention claimed is:
 1. A system for monitoring reactions within aplurality of receptacle vessels comprising: an incubator having atemperature-controlled chamber; a movable receptacle carrier disposedwithin the temperature-controlled chamber and configured to carry aplurality of receptacle vessels and to move the receptacle vesselswithin the temperature-controlled chamber; one or more fixedfluorometers configured to measure a fluorescent emission and positionedwith respect to the receptacle carrier to measure fluorescent emissionsfrom receptacle vessels carried on the receptacle carrier into anoperative position with respect to each fluorometer; one or morefluorescent reference standards mounted on the receptacle carrier; and acontroller configured to control operation of the receptacle carrier andthe one or more fluorometers and to: move the receptacle carrier withrespect to the one or more fluorometers to place a receptacle vesselinto an operative position with respect to each fluorometer; activateeach fluorometer to measure the fluorescent emission intensity from asample contained in the receptacle vessel that is in the operativeposition with respect to the fluorometer; determine a characteristic ofthe reaction based on the measured fluorescent emission intensity fromthe sample contained in the receptacle vessel; move the receptaclecarrier with respect to the one or more fluorometers to place afluorescent standard into optical communication with at least onefluorometer; activate the fluorometer to measure the fluorescentemission intensity of the fluorescent standard that is in opticalcommunication with the fluorometer; determine a deviation of themeasured fluorescent emission intensity of the fluorescent standard froman expected fluorescent emission intensity; if the deviation exceeds athreshold, generate an error signal; and if the deviation does notexceed a threshold, continue operation of the system.
 2. In aninstrument configured to determine a characteristic of a sample from theintensity of a fluorescent emission from the sample, wherein the sampleis contained in a receptacle vessel that is carried on a movablereceptacle carrier, and the intensity of the fluorescent emission ismeasured by a fluorometer that is fixed with respect to the receptaclecarrier and is constructed and arranged to measure the intensity offluorescent emission from a sample contained in a receptacle vessel thatis moved by the receptacle carrier into a detection zone with respect tothe fluorometer, an automated method for detecting failure ordeteriorated performance of the fluorometer with a fluorescent referencestandard mounted on the receptacle carrier, the method comprising thesteps of: (a) moving the receptacle carrier with respect to thefluorometer to periodically place the receptacle vessel into thedetection zone of the fluorometer, and taking one or more measurementsof the intensity of the fluorescent emission from the sample containedin the receptacle vessel with the fluorometer; (b) before or afterperforming step (a), moving the receptacle carrier with respect to thefluorometer to place the fluorescent reference standard into opticalcommunication with the fluorometer, and taking a test measurement of theemission intensity of the fluorescent reference standard; (c)determining a deviation of the test measurement taken in step (b) from apredetermined baseline emission intensity of the fluorescent referencestandard; (d) if the deviation determined in step (c) exceeds athreshold, generate an error signal; and (e) if the deviation determinedin step (c) does not exceed the threshold, continue operation of theinstrument by repeating steps (a) through (c) until a stop condition isreached.
 3. The method of claim 2, further comprising the step ofdetermining the baseline emission intensity by: prior to step (a),moving the receptacle carrier with respect to the fluorometer to placethe fluorescent reference standard into optical communication with thefluorometer; taking an initial measurement of the emission intensity ofthe fluorescent reference standard with the fluorometer; and storing theinitial measurement as the predetermined baseline emission intensity. 4.The system of claim 1, wherein the fluorescent reference standard is afluorescent plastic.
 5. The system of claim 4, wherein the fluorescentreference standard is in the shape of a disk.
 6. The system of claim 1,wherein the fluorescent reference standard is situated beneath areaction vessel.
 7. The system of claim 1, wherein the fluorescentreference standard is out of focus relative to the fluorometer.
 8. Thesystem of claim 1, wherein the fluorescent reference standard is infocus relative to the fluorometer.
 9. The method of claim 2, wherein thefluorescent reference standard is a fluorescent plastic.
 10. The methodof claim 9, wherein the fluorescent reference standard is in the shapeof a disk.
 11. The method of claim 2, wherein the fluorescent referencestandard is situated beneath a reaction vessel.
 12. The method of claim2, wherein the fluorescent reference standard is out of focus relativeto the fluorometer.
 13. The method of claim 2, wherein the fluorescentreference standard is in focus relative to the fluorometer.
 14. Themethod of claim 3, wherein the fluorescent reference standard is afluorescent plastic.
 15. The method of claim 14, wherein the fluorescentreference standard is in the shape of a disk.
 16. The method of claim 3,wherein the fluorescent reference standard is situated beneath areaction vessel.
 17. The method of claim 3, wherein the fluorescentreference standard is out of focus relative to the fluorometer.
 18. Themethod of claim 3, wherein the fluorescent reference standard is infocus relative to the fluorometer.